Histone methylation regulates chromatin structure, transcription, and epigenetic state of the cell. Histone methylation is dynamically regulated by histone methylases and demethylases such as LSD1 and JHDM1, which mediate demethylation of di- and monomethylated histones. It has been unclear whether demethylases exist that reverse lysine trimethylation. We show the JmjC domain-containing protein JMJD2A reversed trimethylated H3-K9/K36 to di- but not mono- or unmethylated products. Overexpression of JMJD2A but not a catalytically inactive mutant reduced H3-K9/K36 trimethylation levels in cultured cells. In contrast, RNAi depletion of the C. elegans JMJD2A homolog resulted in an increase in general H3-K9Me3 and localized H3-K36Me3 levels on meiotic chromosomes and triggered p53-dependent germline apoptosis. Additionally, other human JMJD2 subfamily members also functioned as trimethylation-specific demethylases, converting H3-K9Me3 to H3-K9Me2 and H3-K9Me1, respectively. Our finding that this family of demethylases generates different methylated states at the same lysine residue provides a mechanism for fine-tuning histone methylation.

Here we probe the relationships between assembly of the synaptonemal complex (SC) and progression of recombination between homologous chromosomes during Caenorhabditis elegans meiosis. We identify SYP-2 as a structural component of the SC central region and show that central region assembly depends on proper morphogenesis of chromosome axes. We find that the SC central region is dispensable for initiation of recombination and for loading of DNA strand-exchange protein RAD-51, despite the fact that extensive RAD-51 loading normally occurs in the context of assembled SC. Further, persistence of RAD-51 foci and absence of crossover products in meiotic mutants suggests that SC central region components and recombination proteins MSH-4 and MSH-5 are required to promote conversion of resected double-strand breaks into stable post-strand exchange intermediates. Our data also suggest that early prophase barriers to utilization of sister chromatids as repair templates do not depend on central region assembly.

SummaryHuman neurons are functional over an entire lifetime, yet the mechanisms that preserve function and protect against neurodegeneration during aging are unknown. Here we show that induction of the repressor element 1-silencing transcription/neuron-restrictive silencer factor (REST/NRSF) is a universal feature of normal aging in human cortical and hippocampal neurons. REST is lost, however, in mild cognitive impairment (MCI) and Alzheimer’s disease (AD). Chromatin immunoprecipitation with deep sequencing (ChIP-seq) and expression analysis show that REST represses genes that promote cell death and AD pathology, and induces the expression of stress response genes. Moreover, REST potently protects neurons from oxidative stress and amyloid β-protein (Aβ) toxicity, and conditional deletion of REST in the mouse brain leads to age-related neurodegeneration. A functional ortholog of REST, C. elegans SPR-4, also protects against oxidative stress and Aβ toxicity. During normal aging, REST is induced in part by cell non-autonomous Wnt signaling. However, in AD, frontotemporal dementia and dementia with Lewy bodies, REST is lost from the nucleus and appears in autophagosomes together with pathologic misfolded proteins. Finally, REST levels during aging are closely correlated with cognitive preservation and longevity. Thus, the activation state of REST may distinguish neuroprotection from neurodegeneration in the aging brain.

CRISPR-Cas systems have been used with single-guide RNAs for accurate gene disruption and conversion in multiple biological systems. Here we report the use of the endonuclease Cas9 to target genomic sequences in the C. elegans germline, utilizing single-guide RNAs that are expressed from a U6 small nuclear RNA promoter. Our results demonstrate that targeted, heritable genetic alterations can be achieved in C. elegans, providing a convenient and effective approach for generating loss-of-function mutants.

Analysis of Caenorhabditis elegans syp-1 mutants reveals that both synapsis-dependent and -independent mechanisms contribute to stable, productive alignment of homologous chromosomes during meiotic prophase. Early prophase nuclei undergo normal reorganization in syp-1 mutants, and chromosomes initially pair. However, the polarized nuclear organization characteristic of early prophase persists for a prolonged period, and homologs dissociate prematurely; furthermore, the synaptonemal complex (SC) is absent. The predicted structure of SYP-1, its localization at the interface between intimately paired, lengthwise-aligned pachytene homologs, and its kinetics of localization with chromosomes indicate that SYP-1 is an SC structural component. A severe reduction in crossing over together with evidence for accumulated recombination intermediates in syp-1 mutants indicate that initial pairing is not sufficient for completion of exchange and implicates the SC in promoting crossover recombination. Persistence of polarized nuclear organization in syp-1 mutants suggests that SC polymerization may provide a motive force or signal that drives redispersal of chromosomes. Whereas our analysis suggests that the SC is required to stabilize pairing along the entire lengths of chromosomes, striking differences in peak pairing levels for opposite ends of chromosomes in syp-1 mutants reveal the existence of an additional mechanism that can promote local stabilization of pairing, independent of synapsis. At the onset of meiosis, an extensive spatial reorganization of chromosomes within the nucleus culminates in an arrangement in which homologous chromosomes are lengthwise-aligned, intimately paired, and capable of undergoing crossover recombination. For nearly all sexually reproducing organisms, the accuracy of chromosome segregation during meiosis I depends on the physical exchange between DNA molecules of homologous chromosomes provided by crossover recombination events that are completed in this context. In conjunction with sister-chromatid cohesion, crossing over results in the formation of chiasmata, which serve as mechanical connections that facilitate proper orientation and subsequent segregation of homologs toward opposite poles of the meiosis I spindle (Roeder 1997;Zickler and Kleckner 1999).Specific associations between homologs are established early in meiotic prophase and are maintained prior to and during completion of crossover recombination and chiasma formation. Initial pairing events occur soon after premeiotic S phase, and are typically accompanied by an overall spatial reorganization of the nucleus that leads to a striking polarized distribution of chromosomes and other nuclear contents (for reviews, see Zickler and Kleckner 1998;Scherthan 2001). Many or all aspects of early prophase polarization are lost upon entry into the pachytene stage of meiotic prophase, as homologous chromosomes achieve full intimate alignment with one another along their entire lengths. A hallmark feature of pachytene chromosome organization ...

Many proteins that respond to DNA damage are recruited to DNA lesions. We used a proteomics approach that coupled isotopic labeling with chromatin fractionation and mass spectrometry to uncover proteins that associate with damaged DNA, many of which are involved in DNA repair or nucleolar function. We show that polycomb group members are recruited by poly(ADP ribose) polymerase (PARP) to DNA lesions following UV laser microirradiation. Loss of polycomb components results in IR sensitivity of mammalian cells and Caenorhabditis elegans. PARP also recruits two components of the repressive nucleosome remodeling and deacetylase (NuRD) complex, chromodomain helicase DNA-binding protein 4 (CHD4) and metastasis associated 1 (MTA1), to DNA lesions. PARP plays a role in removing nascent RNA and elongating RNA polymerase II from sites of DNA damage. We propose that PARP sets up a transient repressive chromatin structure at sites of DNA damage to block transcription and facilitate DNA repair. T he cellular response to DNA damage is initiated by the sensing of structural alterations in DNA that culminates in the activation of phosphoinositide-3-kinase-related protein kinases (PIKKs) that include the ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) kinases (1). With the help of mediators, ATM and ATR subsequently signal downstream to activate effector kinases checkpoint 1 (CHK1) and checkpoint 2 (CHK2), leading to transcriptional induction, cell-cycle arrest, DNA repair, senescence, or apoptosis. This DNA damage response induces the sequential recruitment of an extensive network of proteins to the sites of damage. For example, in response to double-strand breaks (DSBs), ATM phosphorylates histone H2AX adjacent to the break to initiate a H2AX-dependent concentration of proteins involved in the DNA damage response, such as mediator of DNA damage checkpoint protein 1 (MDC1), which recruits additional molecules of the ATM kinase. This recruitment effectively initiates a positive feedback loop that promotes the spread of γH2AX-flanking DSBs (2). Phosphorylation of MDC1 by ATM creates a motif that is recognized by the ubiquitin ligase ring finger 8 (RNF8) (3-6) that, with the help of ring finger 168 (RNF168), catalyzes the formation of lysine 63 (K63)-linked polyubiquitin chains that ultimately recruit the breast cancer 1 (BRCA1) A complex containing receptor-associated protein 80 (RAP80), Abraxas, BRCA1, new component of the BRCA1 A complex (NBA1), and BRCA1/BRCA2-containing complex, subunit 3 (BRCC36) (3-10) as well as p53 binding protein 1 (53BP1) and RAD18 homolog (RAD18) (3-8, 11).Several factors, such as Nijmegen breakage syndrome 1 (NBS1), 53BP1, and BRCA1, are recruited to the sites of damage in an H2AX-independent manner (12). However, these interactions appear to be more transient and may play a role as an initial response to DNA damage that is distinct from the extended association of factors via γH2AX. Several additional pathways also have been shown to direct the recruitment of vario...

Summary The Fanconi Anemia (FA) pathway is responsible for interstrand crosslink repair. At the heart of this pathway is the FANCI-FAND2 (ID) complex, which, upon ubiquitination by the FA core complex, travels to sites of damage to coordinate repair that includes nucleolytic modification of the DNA surrounding the lesion and translesion synthesis. How the ID complex regulates these events is unknown. Here we describe a shRNA screen that led to the identification of two nucleases necessary for crosslink repair, FAN1 and EXDL2. FAN1 co-localizes at sites of DNA damage with the ID complex in a manner dependent on FAN1’s ubiquitin binding domain (UBZ), the ID complex, and monoubiquitination of FANCD2. FAN1 possesses intrinsic 5′-3′ exonuclease activity and endonuclease activity that cleaves nicked and branched structures. We propose that FAN1 is a repair nuclease that is recruited to sites of crosslink damage in part through binding the ubiquitinated ID complex through its UBZ domain.

When gene conversion is initiated by a double-strand break (DSB), any nonhomologous DNA that may be present at the ends must be removed before new DNA synthesis can be initiated. In Saccharomyces cerevisiae, removal of nonhomologous ends depends not only on the nucleotide excision repair endonuclease Rad1͞Rad10 but also on Msh2 and Msh3, two proteins that are required to correct mismatched bp. These proteins have no effect when DSB ends are homologous to the donor, either in the kinetics of recombination or in the proportion of gene conversions associated with crossing-over. A second DSB repair pathway, single-strand annealing also requires Rad1͞Rad10 and Msh2͞Msh3, but reveals a difference in their roles. When the f lanking homologous regions that anneal are 205 bp, the requirement for Msh2͞ Msh3 is as great as for Rad1͞Rad10; but when the annealing partners are 1,170 bp, Msh2͞Msh3 have little effect, while Rad1͞Rad10 are still required. Mismatch repair proteins Msh6, Pms1, and Mlh1 are not required. We suggest Msh2 and Msh3 recognize not only heteroduplex loops and mismatched bp, but also branched DNA structures with a free 3 tail.In Saccharomyces cerevisiae, homologous recombination initiated by double-strand breaks (DSBs) can occur by at least two distinct pathways: gene conversion and single-strand annealing (SSA) (1-6). In both cases, the ends of the DSB are resected by a 5Ј to 3Ј exonuclease to produce long 3Ј ended single-strand tails (7,8). In gene conversion, these tails invade a homologous donor sequence and act as primers of new DNA synthesis. However, for this to occur, any nonhomologous bases at the 3Ј end must be removed, so that the primer end may basepair with the donor template. Similarly, in SSA, complementary strands of homologous regions flanking a DSB can anneal, producing an intermediate that has two nonhomologous 3Ј ended tails that must be removed before new DNA synthesis and ligation can occur. In both instances, removal of nonhomologous tails depends on the Rad1 and Rad10 proteins (9), which have been shown in vitro to cleave 3Ј ended nonhomologous tails and which carry out a related function in nucleotide excision repair (NER) (10-12). Other NER proteins are not required (13).In NER, Rad1͞Rad10 are presumably recruited to the site of DNA damage by other proteins of the NER repair complex. We were interested in whether other proteins were required to attract Rad1͞Rad10 to the sites of strand invasion or strand annealing. One set of proteins that recognize DNA distortions are the mismatch repair proteins, most notably Msh2, which has been shown to bind to mismatched bp (14), heteroduplex loops (14), and Holliday junctions (15). Msh2 has been shown to form heterodimers with Msh6 (16,17) and studies of the specificity of mismatch repair have led to the conclusion that Msh2͞Msh6 primarily recognize and correct single bp mismatches, while Msh2 and Msh3 act to correct heteroduplex DNA containing small loops formed by frameshift mutations (16, 18). All of these mismatch repair events ...