Lourdes Serrano

SIRT1 regulates the histone methyl-transferase SUV39H1 during heterochromatin formation.

In contrast to stably repressive, constitutive heterochromatin and stably active, euchromatin, facultative heterochromatin has the capacity to alternate between repressive and activated states of transcription. As such, it is an instructive source to understand the molecular basis for changes in chromatin structure that correlate with transcriptional status. Sirtuin 1 (SIRT1) and suppressor of variegation 3–9 homologue 1 (SUV39H1) are amongst the enzymes responsible for chromatin modulations associated with facultative heterochromatin formation. SUV39H1 is the principal enzyme responsible for the accumulation of histone H3 containing a tri-methyl group at its lysine 9 position (H3K9me3) in regions of heterochromatin. SIRT1 is an NAD+-dependent deacetylase that targets histone H4 at lysine 16 (refs 3 and 4), and through an unknown mechanism facilitates increased levels of H3K9me3 (ref. 3). Here we show that the mammalian histone methyltransferase SUV39H1 is itself targeted by the histone deacetylase SIRT1 and that SUV39H1 activity is regulated by acetylation at lysine residue 266 in its catalytic SET domain. SIRT1 interacts directly with, recruits and deacetylates SUV39H1, and these activities independently contribute to elevated levels of SUV39H1 activity resulting in increased levels of the H3K9me3 modification. Loss of SIRT1 greatly affects SUV39H1-dependent H3K9me3 and impairs localization of heterochromatin protein 1. These findings demonstrate a functional link between the heterochromatin-related histone methyltransferase SUV39H1 and the histone deacetylase SIRT1.

The tumor suppressor SirT2 regulates cell cycle progression and genome stability by modulating the mitotic deposition of H4K20 methylation

The establishment of the epigenetic mark H4K20me1 (monomethylation of H4K20) by PR-Set7 during G2/M directly impacts S-phase progression and genome stability. However, the mechanisms involved in the regulation of this event are not well understood. Here we show that SirT2 regulates H4K20me1 deposition through the deacetylation of H4K16Ac (acetylation of H4K16) and determines the levels of H4K20me2/3 throughout the cell cycle. SirT2 binds and deacetylates PR-Set7 at K90, modulating its chromatin localization. Consistently, SirT2 depletion significantly reduces PR-Set7 chromatin levels, alters the size and number of PR-Set7 foci, and decreases the overall mitotic deposition of H4K20me1. Upon stress, the interaction between SirT2 and PR-Set7 increases along with the H4K20me1 levels, suggesting a novel mitotic checkpoint mechanism. SirT2 loss in mice induces significant defects associated with defective H4K20me1-3 levels. Accordingly, SirT2-deficient animals exhibit genomic instability and chromosomal aberrations and are prone to tumorigenesis. Our studies suggest that the dynamic cross-talk between the environment and the genome during mitosis determines the fate of the subsequent cell cycle.

Homologous Recombination Conserves DNA Sequence Integrity Throughout the Cell Cycle in Embryonic Stem Cells

The maintenance of genomic integrity is crucial to embryonic stem cells (ESC) considering the potential for propagating undesirable mutations to the resulting somatic and germ cell lineages. Indeed, mouse ESC (mESC) exhibit a significantly lower mutation frequency compared to differentiated cells. This could be due to more effective elimination of genetically damaged cells via apoptosis, or especially robust, sequence-conserving DNA damage repair mechanisms such as homologous recombination (HR). We used fluorescence microscopy and 3-dimensional image analysis to compare mESC and differentiated cells, with regard to HR-mediated repair of spontaneous and X-ray-induced double-strand breaks (DSBs). Microscopic analysis of repair foci, flow cytometry, and functional assays of the major DSB repair pathways indicate that HR is greater in mESC compared to fibroblasts. Strikingly, HR appears to be the predominant pathway choice to repair induced or spontaneous DNA damage throughout the ESC cycle in contrast to fibroblasts, where it is restricted to replicated chromatin. This suggests that alternative templates, such as homologous chromosomes, are more frequently used to repair DSB in ESC. Relatively frequent HR utilizing homolog chromosome sequences preserves genome integrity in ESC and has distinctive and important genetic consequences to subsequent somatic and germ cell lineages.

SIRT7 promotes genome integrity and modulates non-homologous end joining DNA repair.

Sirtuins, a family of protein deacetylases, promote cellular homeostasis by mediating communication between cells and environment. The enzymatic activity of the mammalian sirtuin SIRT7 targets acetylated lysine in the N‐terminal tail of histone H3 (H3K18Ac), thus modulating chromatin structure and transcriptional competency. SIRT7 deletion is associated with reduced lifespan in mice through unknown mechanisms. Here, we show that SirT7‐knockout mice suffer from partial embryonic lethality and a progeroid‐like phenotype. Consistently, SIRT7‐deficient cells display increased replication stress and impaired DNA repair. SIRT7 is recruited in a PARP1‐dependent manner to sites of DNA damage, where it modulates H3K18Ac levels. H3K18Ac in turn affects recruitment of the damage response factor 53BP1 to DNA double‐strand breaks (DSBs), thereby influencing the efficiency of non‐homologous end joining (NHEJ). These results reveal a direct role for SIRT7 in DSB repair and establish a functional link between SIRT7‐mediated H3K18 deacetylation and the maintenance of genome integrity.

MiR-93 Controls Adiposity via Inhibition of Sirt7 and Tbx3

Conquering obesity has become a major socioeconomic challenge. Here, we show that reduced expression of the miR-25-93-106b cluster, or miR-93 alone, increases fat mass and, subsequently, insulin resistance. Mechanistically, we discovered an intricate interplay between enhanced adipocyte precursor turnover and increased adipogenesis. First, miR-93 controls Tbx3, thereby limiting self-renewal in early adipocyte precursors. Second, miR-93 inhibits the metabolic target Sirt7, which we identified as a major driver of in vivo adipogenesis via induction of differentiation and maturation of early adipocyte precursors. Using mouse parabiosis, obesity in mir-25-93-106b(-/-) mice could be rescued by restoring levels of circulating miRNA and subsequent inhibition of Tbx3 and Sirt7. Downregulation of miR-93 also occurred in obese ob/ob mice, and this phenocopy of mir-25-93-106b(-/-) was partially reversible with injection of miR-93 mimics. Our data establish miR-93 as a negative regulator of adipogenesis and a potential therapeutic option for obesity and the metabolic syndrome.

Depletion of DSS1 protein disables homologous recombinational repair in human cells.

DSS1 is a small, highly acidic protein widely conserved among eukaryotes as a component of the 19S proteasome and implicated in ubiquitin-mediated proteolysis. The BRCA2 tumor suppressor protein functions in homologous recombinational repair (HRR) of DNA double-strand breaks, and does so in part through the actions of a carboxy-proximal region that binds DNA and several other proteins, including DSS1. In the unicellular eukaryote Ustilago maydis, Dss1 interacts with Brh2, a BRCA2-like protein, and regulates its function in mediating HRR. We used RNA interference to deplete DSS1 in human cells, and assayed the effects on double-strand break repair by homologous recombination. Partial depletion of DSS1 protein in human cells reduced the efficiency of HRR to small fractions of normal levels. Residual HRR activity correlated roughly with the residual level of DSS1 expression. The results imply that mammalian DSS1 makes a critical contribution to the function of BRCA2 in mediating HRR, and hence to genomic stability. Activity of the ubiquitin-proteasome system can influence HRR. However, treatment with proteasome inhibitors only partially reproduced the effects of DSS1 depletion on HRR, suggesting that the function of DSS1 in HRR involves more than proteolysis per se.

Chromatin structure, pluripotency and differentiation:

The state of cell differentiation in adult tissues was once thought to be permanent and irreversible. Since Dollys cloning and, more recently, the generation of induced pluripotent stem cells (iPSCs) from differentiated cells, the traditional paradigm of cell identity has been reexamined. Much effort has been directed toward understanding how cellular identity is achieved and maintained, and studies are ongoing to investigate how cellular identity can be changed. Cell-specific transcription patterns can be altered by modulating the expression of a few transcription factors, which are known as master regulators of cell fate. Epigenetics also plays a major role in cell type specification because the differentiation process is accompanied by major chromatin remodeling. Moreover, whole-genome analyses reveal that nuclear architecture, as defined by the establishment of chromatin domains, regulates gene interactions in a cell-type-specific manner. In this paper, we review the current knowledge of chromatin states that are relevant to both pluripotency and gene expression during differentiation. Information about the epigenetic regulation of gene expression in iPSCs or naïve embryonic stem cells, compared with their differentiated derivatives, will be important as a practical consideration in the long-term maintenance of pluripotent cell cultures for therapeutic purposes.

53BP1 ablation rescues genomic instability in mice expressing ‘RING‐less’ BRCA1

BRCA1 mutations strongly predispose affected individuals to breast and ovarian cancer, but the mechanism by which BRCA1 acts as a tumor suppressor is not fully understood. Homozygous deletion of exon 2 of the mouse Brca1 gene normally causes embryonic lethality, but we show that exon 2‐deleted alleles of Brca1 are expressed as a mutant isoform that lacks the N‐terminal RING domain. This “RING‐less” BRCA1 protein is stable and efficiently recruited to the sites of DNA damage. Surprisingly, robust RAD51 foci form in cells expressing RING‐less BRCA1 in response to DNA damage, but the cells nonetheless display the substantial genomic instability. Genomic instability can be rescued by the deletion of Trp53bp1, which encodes the DNA damage response factor 53BP1, and mice expressing RING‐less BRCA1 do not show an increased susceptibility to tumors in the absence of 53BP1. Genomic instability in cells expressing RING‐less BRCA1 correlates with the loss of BARD1 and a defect in restart of replication forks after hydroxyurea treatment, suggesting a role of BRCA1–BARD1 in genomic integrity that is independent of RAD51 loading.

Chromatin Profiling Reveals Regulatory Network Shifts and a Protective Role for Hepatocyte Nuclear Factor 4α during Colitis

ABSTRACT Transcriptional regulatory mechanisms likely contribute to the etiology of inflammatory bowel disease (IBD), as genetic variants associated with the disease are disproportionately found at regulatory elements. However, the transcription factors regulating colonic inflammation are unclear. To identify these transcription factors, we mapped epigenomic changes in the colonic epithelium upon inflammation. Epigenetic marks at transcriptional regulatory elements responded dynamically to inflammation and indicated a shift in epithelial transcriptional factor networks. Active enhancer chromatin structure at regulatory regions bound by the transcription factor hepatocyte nuclear factor 4α (HNF4A) was reduced during colitis. In agreement, upon an inflammatory stimulus, HNF4A was downregulated and showed a reduced ability to bind chromatin. Genetic variants that confer a predisposition to IBD map to HNF4A binding sites in the human colon cell line CaCo2, suggesting impaired HNF4A binding could underlie genetic susceptibility to IBD. Despite reduced HNF4A binding during inflammation, a temporal knockout model revealed HNF4A still actively protects against inflammatory phenotypes and promotes immune regulatory gene expression in the inflamed colonic epithelium. These findings highlight the potential for HNF4A agonists as IBD therapeutics.

Mammalian HP1 isoforms have specific roles in heterochromatin structure and organization

HP1 is a structural component of heterochromatin. Mammalian HP1 isoforms HP1α, HP1β, and HP1γ play different roles in genome stability, but their precise role in heterochromatin structure is unclear. Analysis of Hp1α-/-, Hp1β-/-, and Hp1γ-/- MEFs show that HP1 proteins have both redundant and unique functions within pericentric heterochromatin (PCH) and also act globally throughout the genome. HP1α confines H4K20me3 and H3K27me3 to regions within PCH, while its absence results in a global hyper-compaction of chromatin associated with a specific pattern of mitotic defects. In contrast, HP1β is functionally associated with Suv4-20h2 and H4K20me3, and its loss induces global chromatin decompaction and an abnormal enrichment of CTCF in PCH and other genomic regions. Our work provides insight into the roles of HP1 proteins in heterochromatin structure and genome stability.