Transcription Of Satellite Repeats Research Articles

Dicer promotes genome stability via the bromodomain transcriptional co-activator BRD4

RNA interference is required for post-transcriptional silencing, but also has additional roles in transcriptional silencing of centromeres and genome stability. However, these roles have been controversial in mammals. Strikingly, we found that Dicer-deficient embryonic stem cells have strong proliferation and chromosome segregation defects as well as increased transcription of centromeric satellite repeats, which triggers the interferon response. We conducted a CRISPR-Cas9 genetic screen to restore viability and identified transcriptional activators, histone H3K9 methyltransferases, and chromosome segregation factors as suppressors, resembling Dicer suppressors identified in independent screens in fission yeast. The strongest suppressors were mutations in the transcriptional co-activator Brd4, which reversed the strand-specific transcription of major satellite repeats suppressing the interferon response, and in the histone acetyltransferase Elp3. We show that identical mutations in the second bromodomain of Brd4 rescue Dicer-dependent silencing and chromosome segregation defects in both mammalian cells and fission yeast. This remarkable conservation demonstrates that RNA interference has an ancient role in transcriptional silencing and in particular of satellite repeats, which is essential for cell cycle progression and proper chromosome segregation. Our results have pharmacological implications for cancer and autoimmune diseases characterized by unregulated transcription of satellite repeats.

M6A RNA methylation of major satellite repeat transcripts facilitates chromatin association and RNA:DNA hybrid formation in mouse heterochromatin.

Heterochromatin has essential functions in maintaining chromosome structure, in protecting genome integrity and in stabilizing gene expression programs. Heterochromatin is often nucleated by underlying DNA repeat sequences, such as major satellite repeats (MSR) and long interspersed nuclear elements (LINE). In order to establish heterochromatin, MSR and LINE elements need to be transcriptionally competent and generate non-coding repeat RNA that remain chromatin associated. We explored whether these heterochromatic RNA, similar to DNA and histones, may be methylated, particularly for 5-methylcytosine (5mC) or methyl-6-adenosine (m6A). Our analysis in mouse ES cells identifies only background level of 5mC but significant enrichment for m6A on heterochromatic RNA. Moreover, MSR transcripts are a novel target for m6A RNA modification, and their m6A RNA enrichment is decreased in ES cells that are mutant for Mettl3 or Mettl14, which encode components of a central RNA methyltransferase complex. Importantly, MSR transcripts that are partially deficient in m6A RNA methylation display impaired chromatin association and have a reduced potential to form RNA:DNA hybrids. We propose that m6A modification of MSR RNA will enhance the functions of MSR repeat transcripts to stabilize mouse heterochromatin.

DICER regulates the expression of major satellite repeat transcripts and meiotic chromosome segregation during spermatogenesis

Constitutive heterochromatin at the pericentric regions of chromosomes undergoes dynamic changes in its epigenetic and spatial organization during spermatogenesis. Accurate control of pericentric heterochromatin is required for meiotic cell divisions and production of fertile and epigenetically intact spermatozoa. In this study, we demonstrate that pericentric heterochromatin is expressed during mouse spermatogenesis to produce major satellite repeat (MSR) transcripts. We show that the endonuclease DICER localizes to the pericentric heterochromatin in the testis. Furthermore, DICER forms complexes with MSR transcripts, and their processing into small RNAs is compromised in Dicer1 knockout mice leading to an elevated level of MSR transcripts in meiotic cells. We also show that defective MSR forward transcript processing in Dicer1 cKO germ cells is accompanied with reduced recruitment of SUV39H2 and H3K9me3 to the pericentric heterochromatin and meiotic chromosome missegregation. Altogether, our results indicate that the physiological role of DICER in maintenance of male fertility extends to the regulation of pericentric heterochromatin through direct targeting of MSR transcripts.

DUX4-induced bidirectional HSATII satellite repeat transcripts form intranuclear double-stranded RNA foci in human cell models of FSHD.

The DUX4 transcription factor is normally expressed in the cleavage-stage embryo and regulates genes involved in embryonic genome activation. Misexpression of DUX4 in skeletal muscle, however, is toxic and causes facioscapulohumeral muscular dystrophy (FSHD). We recently showed DUX4-induced toxicity is due, in part, to the activation of the double-stranded RNA (dsRNA) response pathway and the accumulation of intranuclear dsRNA foci. Here, we determined the composition of DUX4-induced dsRNAs. We found that a subset of DUX4-induced dsRNAs originate from inverted Alu repeats embedded within the introns of DUX4-induced transcripts and from DUX4-induced dsRNA-forming intergenic transcripts enriched for endogenous retroviruses, Alu and LINE-1 elements. However, these repeat classes were also represented in dsRNAs from cells not expressing DUX4. In contrast, pericentric human satellite II (HSATII) repeats formed a class of dsRNA specific to the DUX4 expressing cells. Further investigation revealed that DUX4 can initiate the bidirectional transcription of normally heterochromatin-silenced HSATII repeats. DUX4-induced HSATII RNAs co-localized with DUX4-induced nuclear dsRNA foci and with intranuclear aggregation of EIF4A3 and ADAR1. Finally, gapmer-mediated knockdown of HSATII transcripts depleted DUX4-induced intranuclear ribonucleoprotein aggregates and decreased DUX4-induced cell death, suggesting that HSATII-formed dsRNAs contribute to DUX4 toxicity.

Synergistic lethality between BRCA1 and H3K9me2 loss reflects satellite derepression.

Caenorhabditis elegans has two histone H3 Lys9 methyltransferases, MET-2 (SETDB1 homolog) and SET-25 (G9a/SUV39H1 related). In worms, we found simple repeat sequences primarily marked by H3K9me2, while transposable elements and silent tissue-specific genes bear H3K9me3. RNA sequencing (RNA-seq) in histone methyltransferase (HMT) mutants shows that MET-2-mediated H3K9me2 is necessary for satellite repeat repression, while SET-25 silences a subset of transposable elements and tissue-specific genes through H3K9me3. A genome-wide synthetic lethality screen showed that RNA processing, nuclear RNA degradation, the BRCA1/BARD1 complex, and factors mediating replication stress survival are necessary for germline viability in worms lacking MET-2 but not SET-25. Unlike set-25 mutants, met-2-null worms accumulated satellite repeat transcripts, which form RNA:DNA hybrids on repetitive sequences, additively with the loss of BRCA1 or BARD1. BRCA1/BARD1-mediated H2A ubiquitination and MET-2 deposited H3K9me2 on satellite repeats are partially interdependent, suggesting both that the loss of silencing generates BRCA-recruiting DNA damage and that BRCA1 recruitment by damage helps silence repeats. The artificial induction of MSAT1 transcripts can itself trigger damage-induced germline lethality in a wild-type background, arguing that the synthetic sterility upon BRCA1/BARD1 and H3K9me2 loss is directly linked to the DNA damage provoked by unscheduled satellite repeat transcription.

Contrasting epigenetic states of heterochromatin in the different types of mouse pluripotent stem cells

Mouse embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) represent naive and primed pluripotency states, respectively, and are maintained in vitro by specific signalling pathways. Furthermore, ESCs cultured in serum-free medium with two kinase inhibitors (2i-ESCs) are thought to be the ground naïve pluripotent state. Here, we present a comparative study of the epigenetic and transcriptional states of pericentromeric heterochromatin satellite sequences found in these pluripotent states. We show that 2i-ESCs are distinguished from other pluripotent cells by a prominent enrichment in H3K27me3 and low levels of DNA methylation at pericentromeric heterochromatin. In contrast, serum-containing ESCs exhibit higher levels of major satellite repeat transcription, which is lower in 2i-ESCs and even more repressed in primed EpiSCs. Removal of either DNA methylation or H3K9me3 at PCH in 2i-ESCs leads to enhanced deposition of H3K27me3 with few changes in satellite transcript levels. In contrast, their removal in EpiSCs does not lead to deposition of H3K27me3 but rather removes transcriptional repression. Altogether, our data show that the epigenetic state of PCH is modified during transition from naive to primed pluripotency states towards a more repressive state, which tightly represses the transcription of satellite repeats.

Major satellite repeat RNA stabilize heterochromatin retention of Suv39h enzymes by RNA-nucleosome association and RNA:DNA hybrid formation.

The Suv39h1 and Suv39h2 histone lysine methyltransferases are hallmark enzymes at mammalian heterochromatin. We show here that the mouse Suv39h2 enzyme differs from Suv39h1 by containing an N-terminal basic domain that facilitates retention at mitotic chromatin and provides an additional affinity for major satellite repeat RNA. To analyze an RNA-dependent interaction with chromatin, we purified native nucleosomes from mouse ES cells and detect that Suv39h1 and Suv39h2 exclusively associate with poly-nucleosomes. This association was attenuated upon RNaseH incubation and entirely lost upon RNaseA digestion of native chromatin. Major satellite repeat transcripts remain chromatin-associated and have a secondary structure that favors RNA:DNA hybrid formation. Together, these data reveal an RNA-mediated mechanism for the stable chromatin interaction of the Suv39h KMT and suggest a function for major satellite non-coding RNA in the organization of an RNA-nucleosome scaffold as the underlying structure of mouse heterochromatin.

Let sleeping DNA lie

In hereditary breast and ovarian cancers caused by absence of the BRCA1 protein, genomic instability may ensue from the 'awakening' of repetitive DNA sequences in structurally condensed chromosomal regions. See Article p.179 The BRCA1 protein is a tumour suppressor associated with hereditary breast and ovarian cancer. Inder Verma and colleagues now describe a previously unknown function for BRCA1 that could be relevant to cancer causation. BRCA1 deficiency in mice is shown to impair the integrity of constitutive heterochromatin and to disrupt gene silencing at satellite repeat regions through the loss of ubiquitylation of histone H2A. Abnormal transcription of satellite repeats also occurs in human breast cancer samples deficient in BRCA1, a possible cause of genomic instability and thereby tumorigenesis.

A new role for hth in the early pre-blastodermic divisions in drosophila

In drosophila, the preblastodermic syncytial nuclear divisions occur very fast. In this short period of time chromosomes must condense, segregate and decondense, in conditions governed by maternally provided RNAs and proteins. In this report, we show that the Homothorax (Hth) transcription factor is maternally provided and that its function is necessary for the proper assembly of the centric/centromeric heterochromatin during preblastodermic divisions. Embryos lacking the hth maternally-derived transcript, show abnormal localisation of the centromeric CID protein, and aberrant chromosomal segregation. In this syncytial context, Hth presumably acts together with its partner Extradenticle (Exd) and the RNA PolII, to facilitate transcription of satellite repeats. The transcripts derived from these sequences are needed for the correct assembly of the centric heterochromatin.