Mutations in sterile alpha motif domain and histidine–aspartate domain–containing protein 1 (SAMHD1) are found in a neurodevelopmental disorder, Aicardi–Goutières syndrome, and cancers, and SAMHD1, which is a deoxynucleoside triphosphate (dNTP) triphosphorylase, was identified as a myeloid-specific HIV-1 restriction factor. Here, we characterized the enzymology and structure of an SAMHD1 ortholog of Caenorhabditis elegans, ZK177.8, which also reportedly induces developmental defects upon gene knockdown. We found ZK177.8 protein is a dNTPase allosterically regulated by dGTP. The active site of ZK177.8 recognizes both 2′ OH and triphosphate moieties of dNTPs but not base moiety. The dGTP activator induces the formation of the enzymatically active ZK177.8 tetramers, and ZK177.8 protein lowers cellular dNTP levels in a human monocytic cell line. Finally, ZK177.8 tetramers display very similar X-ray crystal structure with human and mouse SAMHD1s except that its lack of the canonical sterile alpha motif domain. This striking conservation in structure, function, and allosteric regulatory mechanism for the hydrolysis of the DNA building blocks supports their host developmental roles.
RNA polymerase II (Pol II) elongation in metazoans is thought to require phosphorylation of serine 2 (Ser2-P) of the Pol II C-terminal domain (CTD) by the P-TEFb complex, CDK-9/cyclin T. Another Ser2 kinase complex, CDK-12/cyclin K, which requires upstream CDK-9 activity has been identified in Drosophila and human cells. We show that regulation of Ser2-P in C. elegans soma is similar to other metazoan systems, but Ser2-P in the germline is independent of CDK-9, and largely requires only CDK-12. The observed differences are not due to differential tissue expression as both kinases and their cyclin partners are ubiquitously expressed. Surprisingly, loss of CDK-9 from germ cells has little effect on Ser2-P, yet CDK-9 is essential for germline development. By contrast, loss of CDK-12 and Ser2-P specifically from germ cells has little impact on germline development or function, although significant loss of co-transcriptional H3K36 trimethylation is observed. These results show a reduced requirement for Pol II Ser2-P in germline development and suggest that generating Ser2-P is not the essential role of CDK-9 in these cells. Transcriptional elongation in the C. elegans germline thus appears to be uniquely regulated, which may be a novel facet of germline identity.
The proper regulation of spermatogenesis is crucial to ensure the continued production of sperm and fertility. Here, we investigated the function of the H3K4me2 demethylase KDM1A/LSD1 during spermatogenesis in developing and adult mice. Conditional deletion of Kdm1a in the testis just prior to birth leads to fewer spermatogonia and germ cell loss before 3 weeks of age. These results demonstrate that KDM1A is required for spermatogonial differentiation, as well as germ cell survival, in the developing testis. In addition, inducible deletion of Kdm1a in the adult testis results in the abnormal accumulation of meiotic spermatocytes, as well as apoptosis and progressive germ cell loss. These results demonstrate that KDM1A is also required during adult spermatogenesis. Furthermore, without KDM1A, the stem cell factor OCT4 is ectopically maintained in differentiating germ cells. This requirement for KDM1A is similar to what has been observed in other stem cell populations, suggesting a common function. Taken together, we propose that KDM1A is a key regulator of spermatogenesis and germ cell maintenance in the mouse.
The methylation of lysine 4 of Histone H3 (H3K4me) is an important component of epigenetic regulation. H3K4 methylation is a consequence of transcriptional activity, but also has been shown to contribute to “epigenetic memory”; i.e., it can provide a heritable landmark of previous transcriptional activity that may help promote or maintain such activity in subsequent cell descendants or lineages. A number of multi-protein complexes that control the addition of H3K4me have been described in several organisms. These Set1/MLL or COMPASS complexes often share a common subset of conserved proteins, with other components potentially contributing to tissue-specific or developmental regulation of the methyltransferase activity. Here we show that the normal maintenance of H3K4 di- and tri-methylation in the germ line of Caenorhabditis elegans is dependent on homologs of the Set1/MLL complex components WDR-5.1 and RBBP-5. Different methylation states that are each dependent on wdr-5.1 and rbbp-5 require different methyltransferases. In addition, different subsets of conserved Set1/MLL-like complex components appear to be required for H3K4 methylation in germ cells and somatic lineages at different developmental stages. In adult germ cells, mutations in wdr-5.1 or rbbp-5 dramatically affect both germ line stem cell (GSC) population size and proper germ cell development. RNAi knockdown of RNA Polymerase II does not significantly affect the wdr-5.1–dependent maintenance of H3K4 methylation in either early embryos or adult GSCs, suggesting that the mechanism is not obligately coupled to transcription in these cells. A separate, wdr-5.1–independent mode of H3K4 methylation correlates more directly with transcription in the adult germ line and in embryos. Our results indicate that H3K4 methylation in the germline is regulated by a combination of Set1/MLL component-dependent and -independent modes of epigenetic establishment and maintenance.
Background
The processes through which the germline maintains its continuity across generations has long been the focus of biological research. Recent studies have suggested that germline continuity can involve epigenetic regulation, including regulation of histone modifications. However, it is not clear how histone modifications generated in one generation can influence the transcription program and development of germ cells of the next.
Results
We show that the histone H3K36 methyltransferase maternal effect sterile (MES)-4 is an epigenetic modifier that prevents aberrant transcription activity in Caenorhabditis elegans primordial germ cells (PGCs). In mes-4 mutant PGCs, RNA Pol II activation is abnormally regulated and the PGCs degenerate. Genetic and genomewide analyses of MES-4-mediated H3K36 methylation suggest that MES-4 activity can operate independently of ongoing transcription, and may be predominantly responsible for maintenance methylation of H3K36 in germline-expressed loci.
Conclusions
Our data suggest a model in which MES-4 helps to maintain an 'epigenetic memory' of transcription that occurred in germ cells of previous generations, and that MES-4 and its epigenetic product are essential for normal germ cell development.
The hermaphrodite germline of Caenorhabditis elegans initially engages in spermatogenesis and then switches to oogenesis during late stages of larval development. TRA-1, a member of the Ci/Gli family of transcriptional repressors, plays an essential role in this switch by repressing genes that promote spermatogenesis. WDR5 proteins are conserved components of histone methyltransferase complexes normally associated with gene activation. However, two C. elegans WDR5 homologs, wdr-5.1 and wdr-5.2 are redundantly required for normal TRA-1 dependent repression, and this function is independent of their roles in histone methylation. Animals lacking wdr-5.1/wdr-5.2 function fail to switch to oogenesis at 25°C, resulting in a masculinization of germline (Mog) phenotype. The Mog phenotype is caused by ectopic expression of fog-3, a direct target of TRA-1 repression. WDR-5.1 associates with the fog-3 promoter and is required for TRA-1 to bind to fog-3 promoter. Other direct targets of TRA-1 are similarly derepressed in the double mutant. These results show that WDR5 plays a novel and important role in stabilizing transcriptional repression during C. elegans sex determination, and provide evidence that this important protein may operate independently of its established role in histone methyltransferase complexes.
The genetic imprinting of individual loci or whole chromosomes, as in imprinted X-chromosome inactivation in mammals1,2, is established and reset during gametogenesis; defects in this process in the parent can result in disease in the offspring3. We describe a sperm-specific chromatin-based imprinting of the X chromosome in the nematode Caenorhabditis elegans that is restricted to histone H3 modifications. The epigenetic imprint is established during spermatogenesis and its stability in the offspring is affected by the presence of a pairing partner during meiosis in the parental germ line. We observed that DNA lacking a pairing partner during meiosis, the normal situation for the X chromosome in males, is targeted for methylation of histone H3 at Lys9 (H3-Lys9) and can be silenced. Targeting unpaired DNA for silencing during meiosis, a potential hallmark of genome defense, could therefore have a conserved role in imprinted X-chromosome inactivation and, ultimately, in sex chromosome evolution.
Summary
In C. elegans, mRNA production is initially repressed in the embryonic germline by a protein unique to C. elegans germ cells, PIE-1. PIE-1 is degraded upon the birth of the germ cell precursors, Z2 and Z3. We have identified a chromatin-based mechanism that succeeds PIE-1 repression in these cells. A subset of nucleosomal histone modifications, methylated lysine 4 on histone H3 (H3meK4) and acetylated lysine 8 on histone H4 (H4acetylK8), are globally lost and the DNA appears more condensed. This coincides with PIE-1 degradation and requires that germline identity is not disrupted. Drosophila pole cell chromatin also lacks H3meK4, indicating that a unique chromatin architecture is a conserved feature of embryonic germ cells. Regulation of the germline-specific chromatin architecture requires functional nanos activity in both organisms. These results indicate that genome-wide repression via a nanos-regulated, germ cell-specific chromatin organization is a conserved feature of germline maintenance during embryogenesis.
Recently it has been proposed that di-methylation of histone H3 on lysine 4 (H3K4me2) acts as an epigenetic memory to maintain transcriptional patterns in developing tissues. This model suggests that there may be a requirement to reprogram this modification in the germline to prevent transcriptional memory from being inappropriately transmitted to the next generation. We asked if SPR-5, the C. elegans ortholog of the H3K4me2 demethylase LSD1/KDM1, plays a role in epigenetically reprogramming H3K4me2. We show that spr-5 mutants exhibit progressive sterility over many generations due to defects in oogenesis and spermatogenesis. These defects correlate with a progressive failure to erase H3K4me2 in the primordial germ cells, resulting in the misregulation of spermatogenesis-expressed genes due to the transgenerational accumulation of H3K4me2 at these loci. These results suggest that H3K4me2 can serve as an epigenetic memory and that LSD1/KDM1 demethylases play a critical role in the reprogramming of this memory in the germline, preventing inappropriate epigenetic information from being propagated from one generation to the next.
The transition between initiation and productive elongation during RNA Polymerase II (Pol II) transcription is a well-appreciated point of regulation across many eukaryotes. Elongating Pol II is modified by phosphorylation of serine 2 (Ser2) on its carboxy terminal domain (CTD) by two kinases, Bur1/Ctk1 in yeast and Cdk9/Cdk12 in metazoans. Here, we discuss the roles and regulation of these kinases and their relationship to Pol II elongation control, and focus on recent data from work in C. elegans that point out gaps in our current understand of transcription elongation.