H2B monoubiquitination: t'ub or not t'ub for inducible enhancers

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Reference

H2B monoubiquitination: t'ub or not t'ub for inducible enhancers

SEGALA, Grégory, PICARD, Didier

Abstract

Recently, we reported the unexpected finding that the monoubiquitination of histone H2B (H2Bub1) regulates inducible enhancers. Here, we propose a conceptual framework to reconcile the apparently discrepant roles of H2Bub1 in transcription initiation and elongation, and we discuss how H2Bub1 could regulate cellular processes linked to non-coding transcription.

SEGALA, Grégory, PICARD, Didier. H2B monoubiquitination: t'ub or not t'ub for inducible enhancers. Transcription, 2017, vol. 8, no. 2, p. 126-132

DOI : 10.1080/21541264.2017.1285852

Available at:

http://archive-ouverte.unige.ch/unige:93928

Disclaimer: layout of this document may differ from the published version.

H2B monoubiquitination: t’ub or not t’ub for inducible enhancers

Gregory Segala and Didier Picard *

Département de Biologie Cellulaire Université de Genève

Sciences III

30, quai Ernest-Ansermet

CH - 1211 Genève 4, Switzerland

Telephone: +41 22 379 6813

E-mail: didier.picard@unige.ch

* corresponding author

ABSTRACT

Recently, we reported the unexpected finding that the monoubiquitination of histone H2B (H2Bub1) regulates inducible enhancers. Here, we propose a conceptual framework to reconcile the apparently discrepant roles of H2Bub1 in transcription initiation and elongation, and we discuss how H2Bub1 could regulate cellular processes linked to non-coding transcription.

In eukaryotes, controlled access to genetic information by modifications of the

nucleosomes in space and time determines cell phenotype and adapts it to intra- and extracellular variations. Consequently, the nucleosomes are continuously and

dynamically modified to fine-tune the regulation and expression of the genome. The composition of the nucleosome is well conserved across evolution from yeast to mammals, comprising two heterodimers of the core histones H3-H4 together with two heterodimers of the histones H2A-H2B.¹ In addition, some histones can be replaced by their respective histone variants such as H2A.Z for H2A or H3.3 for H3.

Specifically, these two histone variants modify the dynamics of the nucleosome and they are incorporated at the level of regulatory elements such as enhancers and promoters to prime their accessibility to transcription factors.² Histones are modified by a plethora of post-translational modifications that regulate the accessibility of the DNA,³ mark functional elements, control transcription elongation and modulate DNA

replication and repair. In mammals, monoubiquitination of histone H2B on lysine 120 (H2Bub1) is catalyzed by the E3-ubiquitin ligase heterodimer RNF20/RNF40.⁴

H2Bub1 had been mainly characterized as a histone mark related to transcriptional elongation,^5-7 but several other functions have also been attributed to H2Bub1 such as the regulation of RNA processing⁸ or DNA replication.⁹ However, the role of H2B monoubiquitination in gene expression had remained enigmatic¹⁰ because its inhibition only had an effect on a subset of genes¹¹ despite its proposed general involvement in transcription elongation.

H2Bub1 regulates inducible enhancers

Recently, we have characterized a role of H2Bub1 in the control of inducible enhancers.¹² Our initial hypothesis was that H2Bub1 could regulate inducible transcription. This was based on several studies showing that H2Bub1 is important for the regulation of inducible phenotypes like cell differentiation¹³ or inflammation.¹⁴ As a model we used an inducible transcription factor, the estrogen receptor α (ERα), activated as a transcription factor by its cognate ligand 17b-estradiol (E2), and found that H2Bub1 represses the inducible activity of ERα-dependent enhancers. We discovered that H2Bub1 blocks the eviction of the histone variant H2A.Z that is present on inducible enhancer (Fig. 1). Because the dissociation of H2B-H2A or H2B-H2A variant dimers is the first step in nucleosome disassembly,¹ the

stabilization of H2B-H2A.Z dimers by H2Bub1 impairs DNA accessibility upon enhancer induction and is responsible for the repression of inducible transcription.

We provided a mechanism by showing that the monoubiquitination of H2B blocks the

eviction of H2A.Z by impairing the interaction of the chromatin remodeller INO80¹⁵ with H2B.¹²

Constitutive or inducible transcription: a specific H2Bub1 profile?

Previous studies had already reported genomic profiles of H2Bub1,^6,16,17 and claimed that H2Bub1 is restricted to the gene bodies of transcribed genes while H2Bub1 seemed to be at background levels in intergenic regions. In contrast, we focused on the analysis of inducible transcription and enhancers,¹² which are regulatory

elements that are indifferently present in gene bodies as well as in intergenic

regions.¹⁸ As this approach enabled us to find a specific role of H2Bub1 for inducible enhancers, it suggests that some functions of H2Bub1 are restricted to specialized genomic regions. Indeed, in MCF-7 cells, we observed that H2Bub1 is present in the regulatory regions next to the target genes of ERa albeit at lower levels than in gene bodies. Interestingly, treatment with E2 increased H2Bub1 within the gene bodies of ERa target genes, as expected because of the activation of their transcription, but H2Bub1 also increased in regions immediately flanking the ERa-bound enhancers, which are often located in intergenic regions.

MCF-7 cells depend on estrogens for proliferation and as a consequence, they have the ability to extensively remodel their transcriptome when they are induced by E2. It is reasonable to assume that the chromatin of important E2 target genes is primed to respond. We observed that, even before induction by E2, strong ERa-inducible enhancers already display a specific H2Bub1 profile. The priming of chromatin might

be different between constitutive and inducible enhancers and it should definitely be studied for other histone marks as well. Since we exclusively focused on inducible enhancers regulated by steroid receptors, it would be interesting to investigate whether this regulatory mechanism extends to inducible enhancers under the control of other signaling pathways. This appears particularly likely at least for NFkB

considering that it has been demonstrated that inhibition of H2B monoubiquitination increases the recruitment of NFkB on its target genes and their subsequent

activation.¹⁴

H2Bub1 as a buffering histone mark for inducible enhancer?

H2Bub1 is an unusual repressive mark for inducible enhancers since its levels across an inducible enhancer region increase proportionally with the strength of the enhancer (Fig. 1). Since H2Bub1 is a repressor of inducible enhancers, one might expect an opposite correlation between H2Bub1 levels and enhancer strength. To reconcile this paradox, we propose that H2Bub1 might serve to limit the intensity of chromatin remodelling and the resulting DNA exposure. H2Bub1 would be more necessary as enhancer strength increases to prevent excessive opening of

chromatin. The demonstration that H2Bub1 stabilizes nucleosomes¹⁹ fits well with this notion.

H2A.Z has been described as a histone variant that favors the destabilization of the nucleosome.^1,20,21 H2A.Z is incorporated at enhancers and transcription start sites, which are genomic elements that need to be accessible upon activation. H2A.Z was

also found to be associated with the histone variant H3.3 in the same nucleosomes leading to particularly unstable nucleosomes.² H2Bub1 might act as the last safety lock before disassembly of this type of nucleosome. Moreover, we observed that the deposition of H2A.Z on inducible enhancers spreads over several kilobases around the center of these enhancers.¹² Hence, the observed increase of H2Bub1 on both sides of inducible enhancers counteracts the effects of the broader deposition of H2A.Z outside of the enhancers¹² and would prevent the generation of cryptic

transcription initiation sites.²² Improper H2A.Z deposition has indeed previously been associated with cryptic transcription.²³

H2Bub1 valleys at the center of strong inducible enhancers¹² could favor the specific targeting of INO80 to where the DNA must become more accessible for transcription factors (Fig. 1). Upon induction, these H2Bub1 valleys are deepened further,

possibly through the recruitment of deubiquitinases²⁴ at the center of the inducible enhancer, while H2Bub1 increases in the regions flanking it. This mechanism would tend to concentrate the disassembly of nucleosomes at the very center of enhancers while stabilizing nucleosomes around them. As expected, we observed an increase in transcriptional initiation from cryptic sites upon inhibition of H2B

monoubiquitination,¹² further supporting a role for H2Bub1 in spatially restricting chromatin remodelling. The potent roles of H2Bub1 as an intensity and specificity control mark for inducible enhancers could position H2Bub1 as a buffering histone mark that restricts the dynamics of H2A.Z-containing nucleosomes.

Apparent contradictions regarding the proposed role of H2Bub1 in gene activation

It must be acknowledged that the literature appears controversial with respect to the role of H2Bub1 for gene expression.¹⁰ This was even apparent in our own data as we observed that H2Bub1 is associated with active enhancers despite a repressive action on them.¹² Interestingly, a similar apparent contradiction between activation and repressive roles also exists for H2A.Z.^25,26 We demonstrated that H2Bub1 levels correlate with H2A.Z levels,¹² seemingly reinforcing the confusion. Our ChIP-seq data re-analysis highlighted the fact that the stronger an enhancer is, the higher the levels of H2Bub1 are across this enhancer, independently of whether it has already been induced or not. This further suggests that H2Bub1 levels are already set on inducible enhancers before induction, that is at a stage where these enhancers are primed for later activation. This leads us to propose a possible explanation to

reconcile the opposite views on the role of H2Bub1 for transcription. Incorporation of H2A.Z is associated with the priming of the chromatin for enhancer activation.²⁵ Removal of H2Bub1 at the primed enhancer stage may favor the eviction of H2A.Z and consequently may decrease the potential of an enhancer to be activated again later. Upon enhancer activation, H2A.Z has to be removed together with H2B to initiate the disassembly of the nucleosome and to render the DNA accessible for the binding of transcription factors.¹ At this point in time, the stabilization of H2A.Z by H2Bub1 on enhancers inhibits transcription activation. Consequently, this model suggests a time-dependent role for the monoubiquitination of H2B. We cannot exclude that gene-specific effects would influence the kinetics of H2Bub1

modification and chromatin priming. Studies that would specifically address this issue are needed to test the proposed model.

A possible link between H2Bub1 and DNA methylation?

Interestingly, the incorporation of H2A.Z into chromatin is anti-correlated with DNA methylation,²⁷ which itself is linked to the repression of initiation of transcription.²⁸ Moreover, DNA methylation in gene bodies correlates with housekeeping gene expression.²⁹ The main consequence is an absence of H2A.Z from methylated enhancers and promoters of inactive genes, and from genes that are constitutively expressed.^29,30 The reciprocal relationship also exists because H2A.Z incorporation prevents DNA methylation.²⁷ Because H2Bub1 regulates transcription initiation through H2A.Z at inducible enhancers,¹² a link may exist between DNA methylation and H2Bub1 through H2A.Z. Apparently, the monoubiquitination of H2B in gene bodies is not influenced by DNA methylation as H2Bub1 is coupled to the elongation rate of the RNA polymerase II.⁷ However, it is possible that DNA methylation

changes could indirectly affect H2Bub1-related phenotypes by modifying the presence of H2A.Z. Furthermore, DNA methylation and monoubiquitination of H2B could have complementary effects on H2A.Z dynamics by preventing its

incorporation on genes that have to be silenced and by preventing its eviction at cryptic sites close to activated genes, respectively. Possible functional interactions between DNA methylation and H2B monoubiquitination might exist, especially in the context of the specification of constitutive versus inducible transcription, and could be the topic of interesting studies.

Mapping non-coding transcription as a tool to study H2Bub1

Most of the transcription is non-coding³¹ suggesting that many processes associated with transcription elongation could be linked to regulatory mechanisms rather than to the production of coding RNAs. This includes enhancer RNAs (eRNA), which are synthesized in the immediate vicinity of transcription factor binding sites from both strands.³² eRNA would be involved in organizing the looping between enhancers and promoters through the formation of complexes that involve cohesins.³² In light of our study, the increase of the genomic H2Bub1 signal on both sides of H2Bub1 valleys, which is already observed at the primed stage of inducible enhancers,¹² could be due to eRNA synthesis that occurs on both sides of the enhancer but not at its center.³² This hypothesis is reinforced by the fact that H2B monoubiquitination has been demonstrated to be tightly coupled to transcription elongation.⁷ At the same time, it would suggest that prior induction of these enhancers is responsible as a priming event for future induction. Similarly to what we proposed above for H2A.Z, eRNA synthesis would be involved in the stabilization of H2A.Z-containing nucleosome around inducible enhancers through monoubiquitination of H2B to prevent the spreading of DNA accessibility (Fig. 1 and Fig. 2). Intriguingly, the transcription of enhancers occurs before the deposition of the histone marks H3K4me1 and H3K4me2,³³ and there is evidence that the latter may depend on H2Bub1.³⁴ This correlation further supports our hypothesis that eRNA-coupled H2B

monoubiquitination sets the chromatin landscape of inducible enhancers.

A few years ago, non-coding RNAs that are transcribed from sites of DNA double strand breaks were identified and termed DNA damage response RNAs

(DDRNAs).³⁵ They are involved in the recruitment of DNA damage response

proteins. An additional role of the transcription of the DDRNAs could be to increase the monoubiquitination of H2B around the DNA damaged sites (Fig. 2). In support of this notion, H2Bub1 increases at DNA double-strand breaks in an ATM-dependent manner,³⁶ and it is necessary for the recruitment of DNA damage response proteins.

The coupling of DDRNAs synthesis to an increase of H2Bub1 could improve the DNA damage response. It is possible that the regulatory functions of non-coding RNA transcription is mediated in part by the concomitant H2B monoubiquitination, similarly to what we have demonstrated for inducible enhancers.

Conclusions

Gene expression is mainly controlled by enhancers that regulate the rate of

transcription initiation in time and space.¹⁸ If the involvement of H2Bub1 in the control of enhancers induced by steroid receptors extends to other types of enhancers, it could control many other cellular processes that use specific gene expression programs. It would be interesting to map enhancers according to the levels of

H2Bub1 in a model of cell differentiation. This would show whether and how H2Bub1 interferes with cell differentiation specifically through its effects on enhancers. More generally, this analysis could yield more insights into the mechanisms that involve H2Bub1 in the regulation of developmental processes, extending the previously reported link between H2Bub1 and cell differentiation.¹³ The diagnostic value of H2B

monoubiquitination in cancer is controversial.¹⁰ Indeed, some groups reported that the loss of RNF20 or RNF40 was associated with a progression of cancers to high grade stages,^10,37 while others suggested that RNF20 and RNF40 could be markers of bad prognosis.^10,38 It is possible that different outcomes emerge from the diversity of the transcriptomes that would be controlled by H2Bub1 in cancers. For a given cancer, we could expect that depending on the ratio of relevant tumor suppressor genes and oncogenes that depend on H2Bub1, modifications of H2Bub1 levels might differentially affect the outcome. Mapping H2Bub1 on the active and functionally relevant enhancers of different types of cancers could help to solve this issue.

Abbreviations

DDRNAs, DNA damage response RNAs; E2, 17b-estradiol; ERa, estrogen receptor a; H2Bub1, monoubiquitinated H2B.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

G.S. wishes to dedicate this article to the memory of Marie-Jeanne Segala.

Funding

Work in the DP lab on ERa is supported by a grant from the MEDIC foundation and the Canton de Genève.

ORCID

Didier Picard: http://orcid.org/0000-0001-8816-9668

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Legend to Figure 1 : Genomic profiles of H2Bub1 and H2A.Z across inducible enhancers. The graph shows the genomic profiles of H2Bub1 and H2A.Z across inducible enhancers (adapted from ref. ¹²). Nucleosomes are highly enriched in H2Bub1 and H2A.Z on strong inducible enhancers (bold lines) while being poorly enrichedon weak inducible enhancers (light lines). At the center of strong inducible enhancers, H2Bub1 levels decrease to form valleys. Below these profiles, a

representative scheme of the chromatin on strong inducible enhancers depicts the eviction of the histone dimer H2A.Z/H2B by the chromatin remodeler INO80 only on nucleosomes devoid of H2Bub1.

Legend to Figure 2: H2B monoubiquitination could be coupled to RNA synthesis by RNA polymerase II in a variety of processes. H2B monoubiquitination is catalyzed by a heterodimer constituted of the E3-ubiquitin ligases RNF20 and RNF40.⁴ This monoubiquitinated form of histone H2B (H2Bub1) has previously been demonstrated to be coupled to the RNA polymerase II elongation rate.⁷ We propose three different processes in which H2B monoubiquitination is or could be coupled to RNA synthesis by RNA polymerase II. In transcription initiation, enhancer activation is associated with the synthesis of enhancer RNA (eRNA) by RNA polymerase II.³² High levels of H2Bub1 on the sides of inducible enhancers (see also Fig. 1) could be due to eRNA synthesis. For transcription elongation, it is already well established that H2B

monoubiquitination is coupled to pre-messenger RNA (pre-mRNA) synthesis by RNA polymerase II.⁷ To illustrate further our proposal that H2B monoubiquitination could be coupled to non-coding RNA-associated processes, we arbitrarily chose the DNA damage response. Upon DNA double-strand breakage, non-coding RNAs, called

DNA Damage Response RNAs (DDRNAs), are produced from the break sites.³⁵ As it was also observed that H2B is monoubiquitinated in an ATM-dependent manner close to DNA double-strand breaks,³⁶ H2B monoubiquitination could be coupled to DDRNA synthesis. DNA replication⁹ and RNA processing⁸ are additional processes involving H2B monoubiquitination.

Ubiquitin

X ^H2A.Z/H2B

Enrichment

H2Bub1 H2A.Z H2Bub1 H2A.Z

Inducible Enhancer Strong enhancers:

Weak enhancers:

Transcription factor complex assembly RNA Polymerase II recruitment

Enhancer induction

RNF20 RNF40

Induced enhancer

RNA Pol II

RNA

Pol II RNA

Pol II

TRANSCRIPTION INITIATION

TRANSCRIPTION ELONGATION

NON-CODING RNA-ASSOCIATED PROCESS H2B monoubiquitination

?

Ubiquitin

pre-mRNA

DDRNA

DNA double strand break RNA

Pol II RNA

Pol II eRNA

REPLICATION

RNA PROCESSING

Transcription factor/

Coactivator complex H2A.Z/H2B