Transcription suppression is mediated by the HDAC1-Sin3 complex in Xenopus nucleoplasmic extract.

Modification of histones provides a dynamic mechanism to regulate chromatin structure and access to DNA. Histone acetylation, in particular, plays a prominent role in controlling the interaction between DNA, histones, and other chromatin-associated proteins. Defects in histone acetylation patterns interfere with normal gene expression and underlie a wide range of human diseases. Here, we utilize Xenopus egg extracts to investigate how changes in histone acetylation influence transcription of a defined gene construct. We show that inhibition of histone deacetylase 1 and 2 (HDAC1/2) specifically counteracts transcription suppression by preventing chromatin compaction and deacetylation of histone residues H4K5 and H4K8. Acetylation of these sites supports binding of the chromatin reader and transcription regulator BRD4. We also identify HDAC1 as the primary driver of transcription suppression and show that this activity is mediated through the Sin3 histone deacetylase complex. These findings highlight functional differences between HDAC1 and HDAC2, which are often considered to be functionally redundant, and provide additional molecular context for their activity.

The protein kinase CK2 substrate Jabba modulates lipid metabolism during Drosophila oogenesis.

Lipid metabolism plays a critical role in female reproduction. During oogenesis, maturing oocytes accumulate high levels of neutral lipids that are essential for both energy production and the synthesis of other lipid molecules. Metabolic pathways within the ovary are partially regulated by protein kinases that link metabolic status to oocyte development. Although the functions of several kinases in this process are well established, the roles that many other kinases play in coordinating metabolic state with female germ cell development are unknown. Here, we demonstrate that the catalytic activity of casein kinase 2 (CK2) is essential for Drosophila oogenesis. Using an unbiased biochemical screen that leveraged an unusual catalytic property of the kinase, we identified a novel CK2 substrate in the Drosophila ovary, the lipid droplet-associated protein Jabba. We show that Jabba is essential for modulating ovarian lipid metabolism and for regulating female fertility in the fly. Our findings shed light on a CK2-dependent signaling pathway governing lipid metabolism in the ovary and provide insight into the long-recognized but poorly understood association between energy metabolism and female reproduction.

BRD4 promotes resection and homology-directed repair of DNA double-strand breaks.

Double-strand breaks (DSBs) are one of the most toxic forms of DNA damage and represent a major source of genomic instability. Members of the bromodomain and extra-terminal (BET) protein family are characterized as epigenetic readers that regulate gene expression. However, evidence suggests that BET proteins also play a more direct role in DNA repair. Here, we establish a cell-free system using Xenopus egg extracts to elucidate the gene expression-independent functions of BET proteins in DSB repair. We identify the BET protein BRD4 as a critical regulator of homologous recombination and describe its role in stimulating DNA processing through interactions with the SWI/SNF chromatin remodeling complex and resection machinery. These results establish BRD4 as a multifunctional regulator of chromatin binding that links transcriptional activity and homology-directed repair.

Arginine methylation of BRD4 by PRMT2/4 governs transcription and DNA repair.

BRD4 functions as an epigenetic reader and plays a crucial role in regulating transcription and genome stability. Dysregulation of BRD4 is frequently observed in various human cancers. However, the molecular details of BRD4 regulation remain largely unknown. Here, we report that PRMT2- and PRMT4-mediated arginine methylation is pivotal for BRD4 functions on transcription, DNA repair, and tumor growth. Specifically, PRMT2/4 interacts with and methylates BRD4 at R179, R181, and R183. This arginine methylation selectively controls a transcriptional program by promoting BRD4 recruitment to acetylated histones/chromatin. Moreover, BRD4 arginine methylation is induced by DNA damage and thereby promotes its binding to chromatin for DNA repair. Deficiency in BRD4 arginine methylation significantly suppresses tumor growth and sensitizes cells to BET inhibitors and DNA damaging agents. Therefore, our findings reveal an arginine methylation-dependent regulatory mechanism of BRD4 and highlight targeting PRMT2/4 for better antitumor effect of BET inhibitors and DNA damaging agents.

FANCD2 limits acetaldehyde-induced genomic instability during DNA replication in esophageal keratinocytes.

Individuals with Fanconi anemia (FA), a rare genetic bone marrow failure syndrome, have an increased risk of young-onset head and neck squamous cell carcinomas (SCCs) and esophageal SCC. The FA DNA repair pathway is activated upon DNA damage induced by acetaldehyde, a chief alcohol metabolite and one of the major carcinogens in humans. However, the molecular basis of acetaldehyde-induced genomic instability in SCCs of the head and neck and of the esophagus in FA remains elusive. Here, we report the effects of acetaldehyde on replication stress response in esophageal epithelial cells (keratinocytes). Acetaldehyde-exposed esophageal keratinocytes displayed accumulation of DNA damage foci consisting of 53BP1 and BRCA1. At physiologically relevant concentrations, acetaldehyde activated the ATR-Chk1 pathway, leading to S- and G2/M-phase delay with accumulation of the FA complementation group D2 protein (FANCD2) at the sites of DNA synthesis, suggesting that acetaldehyde impedes replication fork progression. Consistently, depletion of the replication fork protection protein Timeless led to elevated DNA damage upon acetaldehyde exposure. Furthermore, FANCD2 depletion exacerbated replication abnormalities, elevated DNA damage, and led to apoptotic cell death, indicating that FANCD2 prevents acetaldehyde-induced genomic instability in esophageal keratinocytes. These observations contribute to our understanding of the mechanisms that drive genomic instability in FA patients and alcohol-related carcinogenesis, thereby providing a translational implication in the development of more effective therapies for SCCs.

Characterization of long-chain acyl-CoA synthetases which stimulate secretion of fatty acids in green algae Chlamydomonas reinhardtii.

BACKGROUND: Microalgae biofuel has become the most promising renewable energy over the past few years. But limitations still exist because of its high cost. Although, efforts have been made in enhancement of lipid productivity, the major cost problem in harvesting and oil extraction is still intractable. Thus, the idea of fatty acids (FAs) secretion which can massively facilitate algae harvesting and oil extraction was investigated here. RESULTS: The cDNAs of two long-chain acyl-CoA synthetases (LACSs) genes were cloned from Chlamydomonas reinhardtii and named as cracs1 and cracs2. They showed different substrate adaptation in the yeast complementation experiments. Cracs2 could utilize FAs C12:0, C14:0, C16:0, C18:0, C16:1 and C18:1, while crac1 could only utilize substrate C14:0, C16:1 and C18:1. Knockdown of cracs1 and cracs2 in C. reinhardtii resulted in accumulation of intracellular lipids. The total intracellular lipids contents of transgenic algae q-15 (knockdown of cracs1) and p-13 (knockdown of cracs2) were 45 and 55 %, respectively higher than that of cc849. Furthermore, FAs secretion was discovered in both transgenic algae. Secreted FAs can reach 8.19 and 9.66 mg/10(9) cells in q-15 and p-13, respectively. CONCLUSION: These results demonstrated the possibility of FAs secretion by microalgae and may give a new strategy of low-cost oil extraction. According to our findings, we proposed that FAs secretion may also be achieved in other species besides Chlamydomonas reinhardtii by knocking-down cracs genes, which may promote the future industrial application of microalgae biofuels.