Histone H2Bub1 deubiquitylation is essential for mouse development, but does not regulate global RNA polymerase II transcription

Abstract

Co-activator complexes dynamically deposit post-translational modifications (PTMs) on histones, or remove them, to regulate chromatin accessibility and/or to create/erase docking surfaces for proteins that recognize histone PTMs. SAGA (Spt-Ada-Gcn5 Acetyltransferase) is an evolutionary conserved multisubunit co-activator complex with modular organization. The deubiquitylation module (DUB) of mammalian SAGA complex is composed of the ubiquitin-specific protease 22 (USP22) and three adaptor proteins, ATXN7, ATXN7L3 and ENY2, which are all needed for the full activity of the USP22 enzyme to remove monoubiquitin (ub1) from histone H2B. Two additional USP22-related ubiquitin hydrolases (called USP27X or USP51) have been described to form alternative DUBs with ATXN7L3 and ENY2, which can also deubiquitylate H2Bub1. Here we report that USP22 and ATXN7L3 are essential for normal embryonic development of mice, however their requirements are not identical during this process, as Atxn7l3−/− embryos show developmental delay already at embryonic day (E) 7.5, while Usp22−/− embryos are normal at this stage, but die at E14.5. Global histone H2Bub1 levels were only slightly affected in Usp22 null embryos, in contrast H2Bub1 levels were strongly increased in Atxn7l3 null embryos and derived cell lines. Our transcriptomic analyses carried out from wild type and Atxn7l3−/− mouse embryonic stem cells (mESCs), or primary mouse embryonic fibroblasts (MEFs) suggest that the ATXN7L3-related DUB activity regulates only a subset of genes in both cell types. However, the gene sets and the extent of their deregulation were different in mESCs and MEFs. Interestingly, the strong increase of H2Bub1 levels observed in the Atxn7l3−/− mESCs, or Atxn7l3−/− MEFs, does not correlate with the modest changes in RNA Polymerase II (Pol II) occupancy and lack of changes in Pol II elongation observed in the two Atxn7l3−/− cellular systems. These observations together indicate that deubiquitylation of histone H2Bub1 does not directly regulate global Pol II transcription elongation.

SAGA-Dependent Histone H2Bub1 Deubiquitination Is Essential for Cellular Ubiquitin Balance during Embryonic Development

“…In contrast, when Pol II transcription was blocked in both human and mouse cells lacking SAGA DUB activity, H2Bub1 was retained, indicating that the SAGA DUB is responsible for removing ub from H2Bub1 to generate free ub [22]. Our recent results further demonstrate that the impairment of H2Bub1 deubiquitylation does not directly impact transcription initiation and/or elongation, because we observed a massive H2Bub1 retention at almost every expressed gene in both Atxn7l3 −/− mouse embryonic stem cells (mESCs) and MEFs (Figure 2), but in contrast, Pol II and Pol II-Ser2P occupancies were only slightly impacted, and only limited subsets of genes changed expression in both cellular systems [34]. In agreement, in Saccharomyces cerevisiae H2B-K123R mutant (which cannot be ubiquitinated), the expression of only a low number of genes was affected [37], further suggesting that ubiquitination and deubiquitination may not have global transcriptional regulatory functions.…”

“…USP22, USP27X, or USP51 require ATXN7L3 and ENY2 for their full activity and are unable to deubiquitinate H2Bub1 alone. In the absence of ATXN7L3, or ENY2, H2Bub1 accumulates on the genome, indicating that without these proteins, the function of the related USPs (USP22, USP27X and USP51) is compromised [30,[32][33][34]. Since H2Bub1 is associated with all transcribed genes, these related DUB enzymes recognize rapidly and specifically H2Bub1, and recycle ub from all active genes, thus creating an equilibrium between Pol II transcription-dependent ub deposition (see below) and the recycling of ub by the three related DUBs.…”

The related coactivator complexes SAGA and ATAC control embryonic stem cell self-renewal through acetyltransferase-independent mechanisms

“…Inactivation of Atac2, an ATAC-specific subunit, was shown to lead to defects in embryonic development at the post-gastrulation stage (Guelman et al, 2009). Similarly, inactivation of genes encoding catalytic subunits of SAGA, Gcn5, Pcaf, Atxn7l3 or Usp22 resulted in embryonic lethality at stages beyond gastrulation (Bu et al, 2007;Koutelou et al, 2019;Xu et al, 2000;Yamauchi et al, 2000;Wang et al, 2021). These phenotypes do not argue for a crucial role of SAGA or ATAC in the inner cell mass of the blastocyst, from which ESCs are derived.…”

Dynamic tissue-specific H2Bub1 is required for human and mouse cardiogenesis

“…To address this, we used previously published H2Bub1-ChIP-seq data of mESCs and MEFs as well as previously published H2Bub1-ChIP-seq data of undifferentiated human fetal osteoblasts (hFOBs) and differentiated hFOBs 28,59 . We generated metagenes corresponding to MEF-specific genes (extra-cellular matrix (ECM) (n = 130)) and hFOB-specific genes (epidermal growth factor related genes (EGF) (n = 24)), with a median length within the 3 rd quartile (median length is 48 Kb for ECM and 99 Kb for EGF) (Supplemental Data 5).…”

“…Interestingly, increased H2Bub1 levels, due to mutations affecting deubiquitination, result in midembryonic lethality in mice 28 , suggesting that development is sensitive to the level of H2Bub1.…”