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Huang et al. BMC Genomics 2013, 14:912 
http://www.biomedcentral.eom/1 471 -21 64/1 4/91 2 



Genomics 



RESEARCH ARTICLE Open Access 



The nucleosome regulates the usage of 
polyadenylation sites in the human genome 

Huan Huang, Jiao Chen, Hongde Liu and Xiao Sun" 



Abstract 

Background: It has been reported that 3' end processing is coupled to transcription and nucleosome depletion 
near the polyadenylation sites in many species. However, the association between nucleosome occupancy and 
polyadenylation site usage is still unclear. 

Results: By systematic analysis of high-throughput sequencing datasets from the human genome, we found that 
nucleosome occupancy patterns are different around the polyadenylation sites, and that the patterns associate with 
both transcription termination and recognition of polyadenylation sites. Upstream of proximal polyadenylation sites, 
RNA polymerase II accumulated and nucleosomes were better positioned compared with downstream of the sites. 
Highly used proximal polyadenylation sites had higher upstream nucleosome levels and RNA polymerase II accumulation 
than lowly used sites. This suggests that nucleosomes positioned upstream of proximal sites function in the recognition 
of proximal polyadenylation sites and in the preparation for 3' end processing by slowing down transcription speed. 
Both conserved distal polyadenylation sites and constitutive sites showed stronger nucleosome depletion near 
polyadenylation sites and had intrinsically better positioned downstream nucleosomes. Finally, there was a higher 
accumulation of RNA polymerase II downstream of the polyadenylation sites, to guarantee gene transcription 
termination and recognition of the last polyadenylation sites, if previous sites were missed. 

Conclusions: Our study indicates that nucleosome arrays play different roles in the regulation of the usage of 
polyadenylation sites and transcription termination of protein-coding genes, and form a dual pausing model of 
RNA polymerase II in the alternative polyadenylation sites' region, to ensure effective 3' end processing. 



Background 

Formation of the 3 ' end of precursor messenger RNA (pre- 
mRNA) is an essential step in the procedure of eukaryotic 
gene expression. Inappropriate 3 ' end formation of human 
mRNAs can have a tremendous impact on health and 
disease [1,2]; however the molecular mode of action is 
still unknown. 3' End processing involves two tightly 
coupled steps, cleavage and polyadenylation, and requires 
a polyadenylation signal (PAS) and a downstream se- 
quence element (DSE) [3]. Transcription termination is 
triggered following recognition of the polyadenylation 
signal by RNA polymerase II (RNAP II) and subsequent 
pre-mRNA cleavage, which occurs at the polyadenylation 
site (polyA site). Interestingly, it has been shown that 
the strength of the polyA site correlates with efficient 
pausing-dependent termination [4,5]. 



* Correspondence: xsun@seu.edu.cn 

State Key Laboratory of Bioelectronics, School of Biological Science and 
Medical Engineering, Southeast University, Nanjing 210096, China 



Recent evidence has indicated that 3' end processing 
is coupled to transcription and splicing, as well as ter- 
mination [6-8], and that RNAP II plays a critical role in 
coordinating co-transcriptional pre-mRNA processing [9]. 
The largest RNAP II subunit contains a carboxyl-terminal 
domain that appears to couple transcription with histone 
methylation, mRNA splicing and polyadenylation by 
mediating interactions with processing factors [10,11]. 
Rigo et al. (2005) have found that the DNA sequence is 
still tethered to RNAP II when the 3' end is cleaved, 
and that the 3' end processing is fast and efficient when 
coupled to transcription in vitro [12]. Glover-Cutter et al 
(2008) have proposed a dual pausing model, where elong- 
ation arrests near the transcription start site and in the 3 ' 
flank to allow co-transcriptional processing by factors 
recruited to the RNAP II ternary complex [13]. 

Epigenetic modifications, including DNA methylation at 
CpG islands [14] and H3K36 methylation [15], can influ- 
ence utilization of alternative polyA sites. In addition, it 



O© 2013 Huang et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative 
BiolVlGCl C6ntTcll Commons Attribution License (http://creativecommons.Org/licenses/by/2.0), which permits unrestricted use, distribution, and 
reproduction in any medium, provided the original work is properly cited. 



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has been reported that nucleosome occupancy drops 
precipitously near the polyadenylation site in many species 
[16-19]. In human T cells, nucleosome density drops 
dramatically near polyA sites with a canonical PAS, and 
highly used polyA sites have greater nucleosome occupancy 
in the immediate downstream region [20]. Khaladkar et al 
(2011) have suggested that a compacted chromatin 
downstream of the polyA sites can slow down the 
elongating transcription, thus facilitating the folding 
of nascent mRNA in a favorable structure at the polyA 
site during transcription [21]. Ji et al. demonstrated that 
compared with lowly expressed genes, highly expressed 
genes had a lower nucleosome level around the polyA 
sites, indicating that transcriptional activity has an add- 
itional impact on the nucleosome level around the 
polyA sites [22]. Our previous studies indicated that 
the nucleosome level around the polyA sites is related 
to conservative sequence elements (PAS and DSE) [23]. 
Altering nucleosome density may affect both RNAP II 
elongation kinetics and polyadenylation, or the recruit- 
ment of the polyadenylation machinery to the nascent 
transcript [24,25]. 

Over half of the genes in the human genome have 
alternative polyA sites [26]. However, nucleosome regu- 
lation during the 3' end processing is still unclear, and 
a detailed exploration of associations between nucleosome 
occupancy and pre-mRNA 3' end formation has not 
been reported. In the present study, we analyzed the 
nucleosome distribution near the polyA sites by processing 
high-throughput experimental data of the human genome. 
Our results suggest that nucleosomes near polyA sites may 
have various roles in the regulation of the usage of different 
types of polyA sites, e.g., proximal and distal polyA sites, 
constitutive and alternative polyA sites. Moreover, there is a 
dual pausing model of RNAP II in the alternative polyA 
sites' region, to ensure effective 3 ' end processing. 

Results 

Nucleosomes immediately downstream of constitutive 
polyA sites are intrinsically better positioned 

Alternative cleavage and polyadenylation is a widespread 
phenomenon in the human genome. Chromatin structure 
is associated with cleavage and polyadenylation [20-23]. 
In the current study, to address the relationship between 
nucleosome occupancy and polyadenylation, the polyA 
sites were divided into constitutive polyA sites and 
alternative sites (see Methods). If a gene has only one 
polyA site, the site is called a constitutive site. If a gene 
has more than one polyA site, those sites are called 
alternative sites. We analyzed nucleosome distribution 
around constitutive and alternative polyA sites. Consistent 
with previous reports [18-20], nucleosome occupancy 
profiles showed deep troughs near polyA sites, which 
were observed in vivo and in vitro (Figure 1, Additional 



file 1A-B), indicating that this is a general phenomenon, 
independent of the type of cells. The fact that the in vivo 
and in vitro results were consistent suggests that the 
depleted nucleosome is partly attributed to DNA sequence. 
Nevertheless, the relative nucleosome level around con- 
stitutive and alternative sites was different in vivo and 
in vitro. Constitutive polyA sites displayed a significantly 
stronger nucleosome depletion near polyA sites than alter- 
native sites did (p < 1 x 10" 19 , Figure 1A) in vivo but not 
in vitro (Figure IB). In other types of cells, the stronger 
nucleosome depletion near constitutive polyA sites was 
also observed (Additional file 1A-B). The results suggest 
that nucleosome profiles around polyA sites are partly 
determined by the DNA sequence and cellular trans- 
factors, in vivo. 

The height of the nucleosome occupancy peak down- 
stream of constitutive polyA sites was higher than that 
downstream of alternative sites (Figure 1A). We quantified 
the significance of this difference using the F test for com- 
paring the summit position distribution and the Students 
t test for comparing the fuzziness score of nucleosome 
peaks called by the DANPOS algorithm within 300 bp 
upstream and 300 bp downstream of the polyA sites for 
the two classes in Figure 1A (Additional file 2). The results 
showed that nucleosomes downstream of constitutive 
polyA sites were better positioned than those down- 
stream of alternative sites. However, this difference was 
not significant in upstream nucleosomes (F test p = 0.057, 
t test p = 0.059). 

Also, we applied the DNA sequences-base nucleosome 
prediction tool developed by the Segal laboratory [19], 
to predict nucleosome occupancy around polyA sites 
(Additional file 1C). The results showed that the nucleo- 
some peak near constitutive sites is more conspicuous 
than that near alternative sites. These analyses confirmed 
our observations that nucleosomes downstream of cons- 
titutive polyA sites are intrinsically better positioned. 
Meanwhile, DNA sequences of alternative polyA sites 
are more disfavored by nucleosomes than constitutive 
sites. However, in vivo, nucleosome levels are higher, 
suggesting that alternative polyA sites are more affected 
by the in vivo environment and can be adjusted easily. 

The relationship between nucleosome level and the 
usage of constitutive and alternative polyA sites 

To explore the regulatory role of the nucleosome in 
polyA site usage, the relationship between nucleosome 
level and the usage of polyA site was analyzed. In the 
case of constitutive polyA sites, the gene expression level 
in the cell reflects the site usage, i.e., in highly expressed 
genes (RPKM > 10) the site usage is high compared with 
that in lowly expressed genes (RPKM < 0.1). The nucleo- 
some level around a constitutive site negatively correlated 
with the gene expression, and there was a proportional 



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-800 -400 polyA 400 800 -800 -400 polyA 400 800 

Figure 1 Nucleosome distribution around constitutive and alternative polyA sites. (A) Nucleosome occupancy surrounding constitutive 
(con) and alternative (alt) polyA sites across a 2000-bp window in human CD4+ T cells. (B) Nucleosome occupancy surrounding constitutive (con) 
and alternative (alt) polyA sites across a 2000-bp window in vitro. 



relationship between RNAP II occupancy and gene expres- 
sion (Figure 2A-B, Additional file 3A-B). This indicates that 
transcription activation can regulate nucleosome levels 
and that RNAP II occupancy is a determinant of nucleo- 
some dismissing, which is consistent with a study by Ji 



et al [22]. To directly assess whether nucleosome posi- 
tioning depends on gene expression, we analyzed a set 
of genes differentially expressed between CD4+ T cells 
and granulocytes (Additional file 4). The results revealed 
that nucleosome level around polyA sites of the expressed 



Constitutive polyA sites 



4 
3.8 



highly expressed 
lowly expressed 




B 



-800 -400 polyA 400 800 
Constitutive polyA sites 



1.4 
1.2 
1 



o 

Q_ 

O 

o 

2 0.8 



Q_ 

< 



0.6 
0= 0.4 
0.2 



-highly expressed 
lowly expressed 



-800 -400 polyA 400 800 



Alternative polyA sites 



_CD 
CD 

E 
o 

o 

_CD 
O 



4 




- high-usage 


3.8 






3.6 






3.4 




^ow-usa^ 


3.2 












2.8 







D 



-800 -400 polyA 400 800 
Alternative polyA sites 



1.05- 



high-usage 
low-usage 




-800 -400 polyA 400 800 



Figure 2 Relationship among nucleosome, RNAP II occupancy and the usage of polyA sites. (A-B) The nucleosome distribution and RNAP 
II occupancy near constitutive polyA sites of highly expressed and lowly expressed genes in CD4+ T cells (highly expressed genes: RPKM > 10, 
blue curve; lowly expressed genes: RPKM < 0.1, red curve). (C-D) The nucleosome distribution and RNAP II occupancy near the high-usage (blue 
curve) and low-usage (red curve) alternative polyA sites in expressed genes (RPKM > 1) in CD4+ T cells. A high-usage site has the lowest RUD in 
the gene, and a low-usage site has the highest RUD in the gene. 



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genes was lower than that of the unexpressed genes in vivo 
and further confirmed that transcription activation can 
regulate nucleosome levels. In highly expressed genes, the 
reduction in downstream nucleosome levels was smaller 
compared with that in upstream ones, and there was a 
much greater accumulation of RNAP II downstream of 
polyA sites than upstream. Therefore, it is reasonable to 
postulate that a nucleosome downstream of constitutive 
sites is expelled by RNAP II more frequently when a gene 
is highly expressed, and that it is better positioned when a 
gene is unexpressed in vivo. Downstream nucleosomes 
may be a huge barrier for RNAP II to pass and promote 
transcription termination. 

To evaluate alternative polyA site usage, the relative 
usage of downstream polyA site score (RUD) was used. 
Briefly, RUD is the ratio between the density of down- 
stream reads and the density of upstream reads (see 
Methods). Alternative polyA sites with the lowest RUD 
in the gene are highly used in transcription, while those 
with the highest RUD in the gene are lowly used. The 
results showed that the changes in nucleosome levels 
between upstream and downstream nucleosomes were 
disproportional (Figure 2C, Additional file 3C-D). For high- 
usage alternative polyA sites, the downstream nucleosome 
level decreased dramatically and RNAP II occupancy in- 
creased quickly (Figure 2D), which is consistent with the 
constitutive sites. The change in the upstream nucleosome 
levels was negligible compared with that in the downstream 
ones. However, the RNAP II profile greatly fluctuated 
between high-usage alternative polyA sites and low-usage 
sites (Figure 2D). There was greater accumulation of 
RNAP II upstream of low-usage polyA sites, suggesting 
a different regulation of upstream nucleosomes. We 
conjectured that nucleosomes downstream of polyA 
sites are intrinsically well positioned and regulated by 
transcription, and upstream nucleosomes may correlate 
with the usage of alternative polyA sites and transcription 
termination. To explain this phenomenon, we performed 
further experiments on alternative polyA sites, which are 
described in the next section. 

Different nucleosome occupancy patterns around 
proximal and distal polyA sites 

The alternative polyA sites appear in different locations, 
and their usage is not random. It is necessary to further 
explore the role of the nucleosome in the regulation of 
alternative polyadenylation. To this end, alternative polyA 
sites were divided into proximal, distal and in-between 
polyA sites, according to the position of the polyA site 
relative to the transcription start site (TSS) of the gene 
(see Methods). For genes with more than two polyA sites, 
the average distances from the three classes of polyA sites 
to the TSS and to the end of the gene were computed 
(Additional file 5). The results showed that the average 



distance from proximal polyA sites to the TSS of the gene 
is the smallest, the average distance from distal polyA sites 
to the end of the gene is the smallest, and the average 
distances from in-between sites to the TSS and to the 
end of the gene are in between. The differences among 
the average distances from the three classes to the TSS 
and to the end of the gene were far more than the length 
of nucleosome DNA (147 bp). Thus, we think that the 
three classes of polyA sites do not influence each other. 

We observed different nucleosome occupancy patterns 
between different types of alternative polyA sites, especially, 
the proximal and distal sites (Figure 3A). The proximal 
sites had higher upstream nucleosome occupancy than 
downstream nucleosome occupancy. The distal sites had 
better positioned downstream nucleosomes than upstream 
ones, which is similar to the constitutive polyA sites 
in vivo. The in-between sites were in between the two 
and their pattern was similar to that of the proximal 
sites. Compared with distal polyA sites, the nucleosome 
levels near and upstream of proximal polyA sites were 
significantly higher (p < 1 x 10" 11 , Figure 3A), whereas 
nucleosomes downstream of distal polyA sites were 
significantly higher than those downstream of proximal 
ones (p < 1 x 10" 15 , Figure 3A). Interestingly, these pat- 
terns of nucleosome distribution were reproduced in 
different cell types, in vitro and in prediction experiments 
(Figure 3B, Additional file 6), indicating an important 
contribution of nucleotide composition to the nucleosome 
occupancy pattern, suggesting that the nucleosome oc- 
cupancy upstream of proximal sites and downstream of 
distal sites is inherently high. 

Through quantitative analysis of physical properties 
of the nucleosome peak called by the DANPOS algo- 
rithm in CD4+ T cells, we confirmed that nucleosomes 
downstream of distal sites have a more consistent summit 
location and lower fuzziness score than those downstream 
of proximal sites (Additional file 2). Therefore, we con- 
cluded that nucleosomes immediately downstream of distal 
polyA sites are intrinsically better positioned. On the other 
hand, proximal polyA sites had more consistent positioning 
of upstream nucleosomes and higher nucleosome occu- 
pancy, suggesting that there are different patterns of 
nucleosome occupancy around proximal and distal sites, 
which may regulate polyadenylation and transcription 
termination by different mechanisms. 

Different regulation roles of nucleosome occupancy in 
the usage of alternative polyadenylation sites 

To explore the different regulatory effects of nucleosome 
occupancy on alternative polyadenylation and transcrip- 
tion termination, the relationship between the usage of 
proximal, distal and in-between polyA sites and nucleo- 
some occupancy was analyzed using RNA-seq datasets 
for different cells. In expressed genes (RPKM > 1), the 



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-800 -400 polyA 400 800 -800 -400 polyA 400 800 



Figure 3 Different patterns of nucleosome distribution around different alternative polyA sites. (A) Nucleosome occupancy around distal 
(d), in-between (m) and proximal (p) polyA sites in human CD4+ T cells {in vivo). (B) Nucleosome occupancy around distal (d), in-between (m) 
and proximal (p) polyA sites in vitro. 

\ J 



alternative polyA sites were divided into high-usage 
ones, with the lowest RUD in the gene, and low-usage 
ones, with the highest RUD in the gene. In the case of 
high-usage and low-usage distal polyA sites, the results 
showed that the varying tendencies of upstream and 
downstream nucleosome occupancy were the same, though 
the change in the upstream nucleosome level was smaller 
than that in the downstream nucleosome level (upstream 
nucleosome p<lx 10" 3 , downstream nucleosome p<lx 
10" 19 ; Figure 4E). The results also indicated that the 
varying tendencies of nucleosome occupancy were similar 
in different cells (Additional file 7A-B). RNAP II accumu- 
lated in the immediate downstream region of high-usage 
distal sites, while the downstream nucleosome level 
was significantly lower compared with the low-usage 
distal sites (Figure 4E-F). This suggests that nucleosome 
occupancy is associated with transcriptional activation. 
These observations are also consistent with constitutive 
polyA sites. Therefore, we concluded that nucleosomes 
downstream of distal polyA sites are similar to those 
downstream of constitutive sites and have a similar 
influence on the regulation the usage of polyA sites and 
transcription termination. 

In proximal polyA sites, the change in downstream 
nucleosome occupancy was in good agreement with the 
change around the distal and constitutive sites, namely, 
the high-usage sites have significantly lower nucleosome 
occupancy than the low-usage sites (Figure 4A, Additional 
file 7C-D). Furthermore, one of the most significant 
findings in our study is that compared with the low- 
usage proximal sites, the high-usage proximal sites had 
a higher upstream nucleosome occupancy (p < 1 x 10" 11 , 
Figure 4A). It was further determined by quantitative 
analysis of nucleosome peak physical properties that 
high-usage proximal polyA sites had better positioned 
upstream nucleosomes (Figure 4A; high-usage Summit 
position = 97.4 ± 61.48, low-usage Summit position = 
105.1 ± 65.75, F test p < 0.01 and Students t test p < 10" 4 ). 



The nucleosome profile of high-usage proximal polyA 
sites shifted downstream compared with that of the 
low-usage sites. Meanwhile, RNAP II accumulated in the 
immediate upstream region of proximal polyA sites, and 
high-usage proximal polyA sites had a higher RNAP II 
occupancy than the low-usage sites had (Figure 4B). 
Furthermore, the RNAP II shifts were largely accom- 
panied by coherent shifts in nucleosome occupancy. In 
in-between polyA sites, the nucleosome level was regu- 
lated by transcription and correlated with the usage of 
polyA sites (Figure 4C). However, the RNAP II occupancy 
was low near in-between polyA sites regardless of polyA 
site usage (Figure 4D). 

These observations imply that proximal polyA sites 
may have better positioned upstream nucleosomes to slow 
down RNAP II speed and improve polyA sites' utilization. 
Figure 5 shows nucleosome-mapping data of two genes 
with high-usage proximal polyA sites or low-usage sites 
in both granulocytes and CD4+ T cells. This mapping 
indicates that nucleosomes downstream of proximal 
polyA sites are intrinsically better positioned and are 
expelled by RNAP II occupancy when the gene is tran- 
scribed, and that nucleosomes upstream of proximal sites 
are adjustable to change in the usage of the proximal sites 
and mark the beginning of transcription termination. 

Discussion 

Alternative polyadenylation is one of the mechanisms in 
human cells that give rise to a variety of transcripts from 
a single gene. More than half of the human genes have 
multiple polyadenylation sites, leading to various mRNA 
and protein products [26]. Epigenetic features and chro- 
matin structure have been shown to be an important 
determinant of polyA site usage [14,15,20]. Here, we 
investigated the patterns of nucleosome distribution 
around different types of polyA sites of protein-coding 
genes in the human genome, and combined mRNA-seq 
and RNAP II datasets to uncover the regulation mechanism 



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CD 
> 
_CD 

CD 
E 

o 

C/) 

o 

_CD 

O 
3 




5' 



-800 -400 polyA 400 800 -800 -400 polyA 400 800 -800 -400 polyA 400 800 



Proximal sites 



In-between sites 



Distal polyA sites 



3' 




-800 -400 polyA 400 800 -800 -400 polyA 400 800 -800 -400 polyA 400 800 

Figure 4 Relationship among nucleosome, RNAP II occupancy and the usage of proximal, in-between and distal polyA sites. (A-B) 

Nucleosome distribution and RNAP II occupancy near high-usage and low-usage proximal polyA sites of expressed genes in CD4+T cell (RPKM > 1). 
(C-D) Nucleosome distribution and RNAP II occupancy near high-usage and low-usage in-between polyA sites of expressed genes in CD4+T cell 
(RPKM > 1). (E-F) Nucleosome distribution and RNAP II occupancy near high-usage and low-usage distal polyA sites of expressed genes in CD4+ T cell 
(RPKM > 1). The blue curve represents high-usage sites that have the lowest RUD in the gene, and the red curve represents low-usage sites that have 
the highest RUD in the gene. 



of nucleosome positioning in alternative polyadenylation 
and transcription termination. Genome-wide mapping of 
nucleosome occupancy in vivo and in vitro revealed that 
different nucleosome occupancy patterns correlate with the 
usage of different polyA sites and transcription termination 
(Summarized in Figure 6). 

The current study demonstrated that compared with 
alternative polyA sites, a constitutive polyA site, defined 
as one polyA site in a gene, had better positioned down- 
stream nucleosomes in vivo and in vitro, which was also 
demonstrated by predicted nucleosome positioning based 
on DNA sequence. Highly expressed genes had lower nu- 
cleosome occupancy near polyA sites than lowly expressed 
genes had. Meanwhile, RNAP II enrichment downstream 
of polyA sites was more pronounced than upstream. 
Thus, our results suggest that constitutive polyA sites 
have a greater intrinsic downstream nucleosome occupancy, 
which affects transcription termination by slowing down 



RNAP II to guarantee recognition of the only polyA site 
and effective transcription termination. 

When examining alternative polyA sites, we found that 
nucleosome occupancy correlates with the sites' position. 
Distal polyA sites, positioned closer to the end of the 
genes, had better positioned downstream nucleosomes 
than other alternative sites both in vivo and in vitro. 
Compared with low-usage distal polyA sites, highly used 
sites had lower nucleosome occupancy and higher RNAP 
II enrichment downstream of the polyA site, which is 
consistent with constitutive polyA sites. On the other 
hand, proximal polyA sites, positioned closer to the TSS 
of the genes, had better positioned upstream nucleo- 
somes. Meanwhile, the pattern of nucleosome occupancy 
around in-between polyA sites was similar to the occu- 
pancy around proximal sites. High-usage proximal sites 
had higher upstream nucleosome occupancy and lower 
downstream nucleosome occupancy. Furthermore, there 



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-900 -600 -300 polyA 300 600 900 -900 -600 -300 polyA 300 600 900 




-900 -600 -300 polyA 300 600 900 -900 -600 -300 polyA 300 600 900 



Figure 5 Nucleosome occupancy surrounding proximal polyA sites of HP1BP3 and TPRG1L genes. HP1BP3 and TPRG1L are highly 
expressed genes in both granulocytes and CD4+ T cells (RPKM > 20). Nucleosome occupancy around the proximal polyA site of HP1BP3 which is 
highly used in both CD4+ T cells (A) and granulocytes (B) (Hs.1 42442.1 .44 chrl - 20975718), and around the proximal polyA site of TPRG1L which 
is lowly used in both CD4+ T cells (C) and granulocytes (D) (Hs.20529.1 .8 chrl + 3535226). 



was a much greater accumulation of RNAP II in the 
high-usage class immediately upstream of the polyA site, 
which is very different from the patterns around distal 
and constitutive sites. We speculated that nucleosomes 
upstream of proximal polyA sites are adjustable, which 
correlates with polyA site usage. 

Constitutive polyA sites can be taken as high-usage 
sites. Distal sites, favored in differentiated cells, exhibit a 
much higher frequency of canonical PAS and canonical 
DSE motif (likely stronger sites), and proximal polyA 
sites, favored in proliferating cells, tend to have limited 
consensus PAS features (likely weaker sites) [27]. We 



also found that distal polyA sites are more favored in 
granulocyte, CD8+ T cells and CD4+ T cells (Additional 
file 8). Thus, our results are in good agreement with a 
previous study showing that downstream nucleosomes 
correlate with the usage of polyA sites [20]. It has been 
shown that highly nucleosome occupancy downstream 
of polyA sites strongly correlated with a more favorable 
mRNA structure and greater accumulation of RNAP II 
at the polyA site [21], and improved the usage frequency 
of polyA sites. Furthermore, Grosso et al. have shown 
that nucleosome occupancy at the 3' end of genes is 
dynamic and correlates with RNAP II density [28]. In 




Figure 6 The dual pausing model of RNAP II and nucleosome occupancy patterns during regulation of the usage of different polyA 
sites and transcription termination. A nucleosome upstream of a proximal site, which is adjustable and related to the site usage, is the first 
barrier pausing RNAP II. Meanwhile, it signals to RNAP II to prepare for a 3' end processing event, by slowing down transcription speed. A 
nucleosome downstream of distal or constitutive polyA site, which is intrinsically well positioned and regulated by transcription, is the second 
barrier pausing RNAP II to ensure transcription termination and recognition of the last polyadenylation sites if previous sites were missed. 



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addition, the results of Additional file 8 and Figure 4B 
can explain the phenomenon shown in Figure 2D. RNAP 
II accumulated immediately upstream of proximal polyA 
sites (Figure 4B). In low-usage alternative sites, proximal 
sites outnumber distal sites; almost triple the percentage 
of distal sites (Additional file 8). Thus, RNAP II accumu- 
lated immediately upstream of low-usage alternative polyA 
sites. Moreover, we observed that there was a little peak in 
the RNAP II profile upstream of high-usage alternative 
polyA sites (Figure 2D), which was attributed to the con- 
tribution of proximal sites of the high-usage alternative 
polyA sites. 

Our results specifically point out that downstream nu- 
cleosomes correlate with the usage of constitutive and 
distal sites. Additionally, we inferred that nucleosomes 
downstream of constitutive and distal polyA sites are 
intrinsically better positioned, to guarantee stronger polyA 
site recognition and facilitate the formation of a stable 
RNA structure at the polyA site by reducing RNAP II 
transcription speed. Moreover, the nucleosome occupancy 
curve around constitutive and distal polyA sites was much 
steeper than other sites in vivo, which makes it more diffi- 
cult for RNAP II to pass. Also, accumulation of RNAP II 
at the 3 ' end of genes correlated with the usage of polyA 
sites. Thus, a possible role of downstream nucleosomes 
is to guarantee gene transcription termination and rec- 
ognition of the last polyadenylation sites if previous 
sites were missed. 

Proximal polyA sites have an intrinsic advantage over 
the distal ones, as they are transcribed earlier and have 
more time to be recognized ("first come, first served"), 
while higher elongation rate leads to them being missed 
and to enhance the recognition of the distal polyA sites 
[29]. According to the mechanisms of alternative polya- 
denylation regulation, the proximal polyA site may mainly 
serve as an alternative site. Hence, we inferred that nucle- 
osomes upstream of polyA sites play regulatory roles 
that promote the recognition of weaker proximal sites 
by slowing down transcription speed and recruiting 3- 
processing factors before RNAP II arrives at the polyA 
site. On the other hand, they may serve as the boundary 
of the polyA sites region, signaling to RNAP II to slow 
down and prepare for a 3' end processing event. Such 
signaling may be necessary to reduce transcription speed 
in polyA + genes, where there is alternative polyadenyla- 
tion. Furthermore, there was a nucleosome shift toward 
the 3 ' end in highly used sites, which was largely accom- 
panied by a coherent shift of RNAP II occupancy. We 
conjecture that this may be related to the dynamic mech- 
anism of transcription through chromatin by RNAP II. 
Chromatin remodeling factors and histone chaperones 
dissociate from the transcription complex near the polyA 
site to help the polymerase remodel the nucleosome [30]. 
The increase in RNAP II residence time near a proximal/ 



distal site may in turn reduce the amount of RNAP II that 
can be captured near in-between sites. 

Conclusions 

Taken together, our study indicates that nucleosomes 
near polyA sites have various regulatory effects on poly- 
adenylation and transcription termination (Figure 6). 
Proximal polyadenylation sites have an adjustable upstream 
nucleosome occupancy, which correlates with the site 
usage, and may signal to RNA polymerase II to slow 
down and prepare for a 3 ' end processing event. Distal 
and constitutive sites have intrinsically well-positioned 
downstream nucleosomes, which are regulated by tran- 
scription and are the second barrier pausing RNAP II 
to ensure transcription termination and recognition of 
the last polyadenylation sites, if previous sites were missed. 
Nucleosome arrays around polyA sites play different roles 
in the regulation of the usage of polyadenylation sites and 
transcription termination of protein-coding genes, and 
form a dual pausing model of RNA polymerase II in the 
alternative polyadenylation sites' region, to ensure effective 
3' end processing. Histone modification as the main epi- 
genetic marker has been shown to regulate alternative 
splicing by affecting splicing regulators and recruiting the 
spliceosome [31]. It will be interesting to examine how 
different types of histone modifications affect different 
types of polyA site usage. 

Methods 

PolyA site dataset 

The genomic coordinates of the polyA sites of the human 
genome were obtained from the PolyA_DB2 database 
[26]. We used the Batch Coordinate Conversion (liftOver) 
tool from the UCSC Genome Bioinformatics resource 
to remap the polyA sites from NCBI35/hgl7 to NCBI36/ 
hgl8 (http://genome.ucsc.edu/cgi-bin/hgLiftOver) [32]. 
All polyA sites that were not uniquely mapped to the 
reference sequence of the protein-coding gene were 
removed. PolyA sites in genes with only one polyA site 
are called constitutive sites. PolyA sites in genes with 
multiple sites were alternative sites. According to the 
number of polyA sites in the gene, genes were divided 
into two classes: 8412 genes with a single constitutive 
polyA site and 9311 genes with alternative polyA sites. 

In genes with alternative polyA sites, the sites were 
divided into proximal, distal and in-between polyA 
sites, according to the position of the polyA site relative 
to the TSS of the gene. The polyA sites closest to the 
TSS of a gene were proximal sites, the sites closest to 
the end of a gene were distal sites, and the polyA sites 
located between proximal and distal polyA sites were 
in-between sites. The coordinates of the TSS and the 
end of genes were obtained from the annotation of 



Huang et al. BMC Genomics 2013, 14:912 
http://www.biomedcentral.eom/1 471 -21 64/1 4/91 2 



Page 9 of 10 



RefSeq genes in UCSC Table Browser (hgl8 database, 
http://genome.ucsc.edu/cgi-bin/hgTables). 

MNase sequencing and RNAP II data and data analysis 

Sequencing datasets of nucleosome positions in different 
human cell types (CD4+ T cells, CD8+ T cells and gran- 
ulocytes) and in vitro, generated by high-throughput 
SOLiD technology, were analyzed [33]. Tag coordinate 
bed files of RNAP II in human CD4+ T cells using the 
Solexa sequencing technology were also used [34] . 

Nucleosome scores calculated as previously described 
[33], represent the average number of sequenced reads 
uniquely mapping to the sense strand 80 bases upstream 
and to the antisense strand 80 bases downstream of one 
locus with a step size of 10 bp. Nucleosome occupancy 
was calculated in alignment with polyA sites, and was 
smoothed using a moving average filter. The moving 
average span was 5. The Wilcoxon rank sum test was 
used to gauge the significance (p). RNAP II occupancy 
was calculated similarly to the nucleosome occupancy, but 
the score represents the average number of sequenced 
reads uniquely mapped to the sense strand 300 bases 
upstream and to the antisense strand 300 bases down- 
stream of one locus. 

Nucleosome occupancy peak calling was performed 
by the DANPOS algorithm [35], which was designed 
for dynamic nucleosome analysis at single-nucleotide 
resolution by sequencing and can transform reads data 
of each replicate to occupancy and provide peak summit 
position and peak fuzziness score. 



Analysis of gene expression levels and polyA site usage 
using mRNA-seq data 

The mRNA-seq data performed on nuclear extracts from 
human CD4+ T cells, CD8+ T cells and granulocytes 
(accession number GSE25133) [33] were obtained from 
NCBIs Gene Expression Omnibus. The sequencing reads 
were aligned using TopHat against human genome (hgl8) 
[36], allowing at most two mismatches. Gene expression 
levels were quantified using read density in the protein- 
coding region based on the reads per kilobase of mappable 
region per million mapped reads (RPKM) method [37]. 

To evaluate polyA site usage, the density of reads mapped 
to upstream and downstream regions was compared, as 
illustrated in a previous study [38]. The relative usage 
of downstream polyA site (RUD) score was calculated, 
which is the ratio between the density of downstream 
reads and the density of upstream reads. A low RUD 
score for a polyA site represents high usage of the polyA 
site. The reads mapped in the ±10-nt region around the 
polyA sites were not used for RUD calculation, because 
usually the cleavage sites are not precise. 



Additional files 



Additional file 1: Nucleosome distribution around constitutive and 
alternative polyA sites. (A-B) Nucleosome occupancy surrounding 
polyA sites of constitutive (con) and alternative (alt) polyA sites across a 
2000-bp window in granulocytes and CD8+ T cells. (C) Predicted 
nucleosome occupancy based on DNA sequence around constitutive 
(con) and alternative (alt) polyA sites across a 2000-bp window. 

Additional file 2: Statistics of the summit position distribution and 
fuzziness score of nucleosome peaks called by the DANPOS 
algorithm within 300 bp upstream and 300 bp downstream of the 
polyA sites. The standard deviation (std) of the distance between the 
summit position and polyA sites, and the average value of fuzziness score 
of nucleosome peaks were calculated to appraise the consistency of 
nucleosome positioning. The sample used for the analyses was CD4+ 
T cells shown in Figures 1A and 3A. 

Additional file 3: The relationship between the nucleosome and the 
usage of polyA sites. (A-B) Nucleosome distribution near constitutive 
polyA sites of highly expressed and lowly expressed genes in granulocytes 
and CD8+T cells (highly expressed genes: RPKM > 10, blue curve; lowly 
expressed genes: RPKM < 0.1, red curve). (C-D) Nucleosome distribution 
near high-usage (blue curve) and low-usage (red curve) alternative polyA 
sites in expressed genes in granulocytes and CD8+ T cells (RPKM > 1). 
High-usage sites have the lowest RUD in the gene, and low-usage sites have 
the highest RUD in the gene. 

Additional file 4: Nucleosome level around constitutive polyA sites of 
differentially expressed genes between CD4+ T cells and granulocytes. 

The blue curve represents the genes that were highly expressed in CD4+ T 
cells (RPKM > 10) and unexpressed in granulocytes (RPKM < 0.1). The red curve 
represents the genes that were unexpressed in CD4+ T cells (RPKM < 0.1) and 
highly expressed in granulocytes (RPKM >10). 

Additional file 5: The average distance from the three classes of 
polyA sites to the TSS and to the end of the gene. 

Additional file 6: Different patterns of nucleosome distribution 
around different alternative polyA sites. (A-B) Nucleosome occupancy 
around distal (d), in-between (m) and proximal (p) polyA sites across a 
2000-bp window in granulocytes and CD8+ T cells. (C) Predicted 
nucleosome occupancy based on DNA sequence around distal (d), 
in-between (m) and proximal (p) polyA sites. 

Additional file 7: Relationship between nucleosomes and the usage 
of proximal and distal polyA sites. (A-B) Nucleosome occupancy 
surrounding high-usage and low-usage distal polyA sites of expressed genes 
(RPKM > 1) in granulocytes and CD8+ T cells. (C-D) Nucleosome occupancy 
surrounding high-usage and low-usage proximal polyA sites of expressed 
genes (RPKM > 1) in granulocytes and CD8+ T cells. The blue curve 
represents high-usage sites that have the lowest RUD in the gene; and the 
red curve represents low-usage sites that have the highest RUD in the gene. 

Additional file 8: Percentage of distal and proximal polyA sites in 
high-usage and low-usage alternative polyA sites. The percentage of 
distal (distal) and proximal (proximal) polyA sites in high-usage and 
low-usage alternative polyA sites in granulocytes (A), CD4+ T cells (B) 
and CD8+ T cells (C). High-usage polyA sites have the lowest RUD in 
expressed genes (RPKM > 1), and low-usage polyA sites have the highest 
RUD in expressed genes (RPKM > 1). 



Competing interests 

The authors declare that they have no competing interests. 
Authors' contributions 

HH conceived the study and wrote the paper. HH, HDL and XS designed the 
bioinformatics analyses. JC collected and preprocessed data sets. All authors 
read and approved the final manuscript. 

Acknowledgements 

This work was supported by a grant from the National Basic Research 
Program of China (No. 2012CB316501), the National Natural Science 
Foundation of China (61073141, 31240080 and 31371339) and the Leading 
Science Project of Southeast University. 



Huang et al. BMC Genomics 201 3, 14:91 2 Page 1 0 of 1 0 

http://www.biomedcentral.eom/1 471 -21 64/1 4/91 2 



Received: 27 April 2013 Accepted: 19 December 2013 
Published: 23 December 2013 



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doi:1 0.1 186/1471-2164-14-912 

Cite this article as: Huang et al.: The nucleosome regulates the usage of 
polyadenylation sites in the human genome. BMC Genomics 2013 14:912. 



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