Concentrating pre-mRNA processing factors in the histone locus body facilitates efficient histone mRNA biogenesis

The histone locus body (HLB) assembles at replication-dependent histone genes and concentrates factors required for histone messenger RNA (mRNA) biosynthesis. FLASH (Flice-associated huge protein) and U7 small nuclear RNP (snRNP) are HLB components that participate in 3′ processing of the nonpolyadenylated histone mRNAs by recruiting the endonuclease CPSF-73 to histone pre-mRNA. Using transgenes to complement a FLASH mutant, we show that distinct domains of FLASH involved in U7 snRNP binding, histone pre-mRNA cleavage, and HLB localization are all required for proper FLASH function in vivo. By genetically manipulating HLB composition using mutations in FLASH, mutations in the HLB assembly factor Mxc, or depletion of the variant histone H2aV, we find that failure to concentrate FLASH and/or U7 snRNP in the HLB impairs histone pre-mRNA processing. This failure results in accumulation of small amounts of polyadenylated histone mRNA and nascent read-through transcripts at the histone locus. Thus, the HLB concentrates FLASH and U7 snRNP, promoting efficient histone mRNA biosynthesis and coupling 3′ end processing with transcription termination

Drosophila Symplekin localizes dynamically to the histone locus body and tricellular junctions

The scaffolding protein Symplekin is part of multiple complexes involved in generating and modifying the 3' end of mRNAs, including cleavage-polyadenylation, histone pre-mRNA processing and cytoplasmic polyadenylation. To study these functions in vivo, we examined the localization of Symplekin during development and generated mutations of the Drosophila Symplekin gene. Mutations in Symplekin that reduce Symplekin protein levels alter the efficiency of both poly A+ and histone mRNA 3' end formation resulting in lethality or sterility. Histone mRNA synthesis takes place at the histone locus body (HLB) and requires a complex composed of Symplekin and several polyadenylation factors that associates with the U7 snRNP. Symplekin is present in the HLB in the early embryo when Cyclin E/Cdk2 is active and histone genes are expressed and is absent from the HLB in cells that have exited the cell cycle. During oogenesis, Symplekin is preferentially localized to HLBs during S-phase in endoreduplicating follicle cells when histone mRNA is synthesized. After the completion of endoreplication, Symplekin accumulates in the cytoplasm, in addition to the nucleoplasm, and localizes to tricellular junctions of the follicle cell epithelium. This localization depends on the RNA binding protein ypsilon schachtel. CPSF-73 and a number of mRNAs are localized at this same site, suggesting that Symplekin participates in cytoplasmic polyadenylation at tricellular junctions

Interrogating the Function of Metazoan Histones using Engineered Gene Clusters

Histones and their post-translational modifications influence the regulation of many DNA-dependent processes. Although an essential role for histone-modifying enzymes in these processes is well established, defining the specific contribution of individual histone residues remains a challenge because many histone-modifying enzymes have non-histone targets. This challenge is exacerbated by the paucity of suitable approaches to genetically engineer histone genes in metazoans. Here, we describe a facile platform in Drosophila for generating and analyzing any desired histone genotype, and we use it to test the in vivo function of three histone residues. We demonstrate that H4K20 is neither essential for DNA replication nor for completion of development, unlike conclusions drawn from analyses of H4K20 methyltransferases. We also show that H3K36 is required for viability and H3K27 is essential for maintenance of cellular identity during development. These findings highlight the power of engineering histones to interrogate genome structure and function in animals

A Gnotobiotic Mouse Model Demonstrates That Dietary Fiber Protects against Colorectal Tumorigenesis in a Microbiota- and Butyrate-Dependent Manner

It is controversial whether dietary fiber protects against colorectal cancer because of conflicting results from human epidemiologic studies. However, these studies and mouse models of colorectal cancer have not controlled the composition of gut microbiota, which ferment fiber into short-chain fatty acids such as butyrate. Butyrate is noteworthy because it has energetic and epigenetic functions in colonocytes and tumorsuppressive properties in colorectal-cancer cell lines. We utilized gnotobiotic mouse models colonized with wild-type or mutant strains of a butyrate-producing bacterium to demonstrate that fiber does have a potent tumor-suppressive effect but in a microbiota- and butyrate-dependent manner. Furthermore, due to the Warburg effect, butyrate was metabolized less in tumors where it accumulated and functioned as an HDAC inhibitor to stimulate histone acetylation and affect apoptosis and cell proliferation. To support the relevance of this mechanism in human cancer, we demonstrate that butyrate and histone-acetylation levels are elevated in colorectal adenocarcinomas compared to normal colonic tissues

Drosophila Symplekin localizes dynamically to the histone locus body and tricellular junctions

The scaffolding protein Symplekin is part of multiple complexes involved in generating and modifying the 3' end of mRNAs, including cleavage-polyadenylation, histone pre-mRNA processing and cytoplasmic polyadenylation. To study these functions in vivo, we examined the localization of Symplekin during development and generated mutations of the Drosophila Symplekin gene. Mutations in Symplekin that reduce Symplekin protein levels alter the efficiency of both poly A(+) and histone mRNA 3' end formation resulting in lethality or sterility. Histone mRNA synthesis takes place at the histone locus body (HLB) and requires a complex composed of Symplekin and several polyadenylation factors that associates with the U7 snRNP. Symplekin is present in the HLB in the early embryo when Cyclin E/Cdk2 is active and histone genes are expressed and is absent from the HLB in cells that have exited the cell cycle. During oogenesis, Symplekin is preferentially localized to HLBs during S-phase in endoreduplicating follicle cells when histone mRNA is synthesized. After the completion of endoreplication, Symplekin accumulates in the cytoplasm, in addition to the nucleoplasm, and localizes to tricellular junctions of the follicle cell epithelium. This localization depends on the RNA binding protein ypsilon schachtel. CPSF-73 and a number of mRNAs are localized at this same site, suggesting that Symplekin participates in cytoplasmic polyadenylation at tricellular junctions

Drosophila Symplekin localizes dynamically to the histone locus body and tricellular junctions

Behavioral problems are multifactorial and includes perinatal, maternal, family, parenting, socio-economic and personal risk factors, but less is known about the association of postnatal heavy metals on children’s behavioral problems in Pacific Island children. Methods: A cohort of eligible nine-year-old children within a Pacific Island Families longitudinal study were recruited for a cross-sectional study. Child behavior problems were assessed using the child behavior checklist. Heavy metals (including Ni, Cu, Pb, Al, Cr and Cd) were determined in toenails, after acid digestion and analyzed using inductively coupled plasma mass spectrometry. Other factors such as lifestyle (smoking in pregnancy), health outcomes (obesity, health status), demographics (gender, ethnicity, parents’ marital status) and socioeconomic status (household income levels) were also collected. The statistical analysis included t-tests for independent sample and Mann–Whitney U-test, and chi-square or Fisher’s exact tests of independence for comparisons of the proportions. Regression models tested the hypothesized risk factors for behavior outcomes. Results: This observational study enrolled 278 eligible Pacific Island children living in Auckland, New Zealand. The prevalence of behavioral problems in the clinical range was high (22%) but there was no significant association between heavy metals in toenails and adverse behavioral outcomes. Conclusion: Regular monitoring and assessments of children for environmental risk factors, as well as social and lifestyle factors for behavior problems, continues. Alternative indicators of exposure to heavy metal should be evaluated

A mixed methods evaluation of family-driven care implementation in juvenile justice agencies in Georgia

Abstract Background Improving family engagement in juvenile justice (JJ) system behavioral health services is a high priority for JJ systems, reform organizations, and family advocacy groups across the United States. Family-driven care (FDC) is a family engagement framework used by youth-serving systems to elevate family voice and decision-making power at all levels of the organization. Key domains of a family-driven system of care include: 1) identifying and involving families in all processes, 2) informing families with accurate, understandable, and transparent information, 3) collaborating with families to make decisions and plan treatments, 4) responding to family diversity and inclusion, 5) partnering with families to make organizational decisions and policy changes, 6) providing opportunities for family peer support, 7) providing logistical support to help families overcome barriers to participation, and 8) addressing family health and functioning. FDC enhances family participation, empowerment, and decision-making power in youth services; ultimately, improving youth and family behavioral health outcomes, enhancing family-child connectedness, and reducing youth recidivism in the JJ setting. Methods We evaluated staff-perceived adoption of the eight domains of FDC across detention and community services agencies in the state of Georgia. We collected mixed methods data involving surveys and in-depth qualitative interviews with JJ system administrators, staff, and practitioners between November 2021- July 2022. In total, 140 individuals from 61 unique JJ agencies participated in surveys; and 16 JJ key informants participated in qualitative interviews. Results FDC domains with the highest perceived adoption across agencies included identifying and involving families, informing families, collaborative decision-making and treatment planning, and family diversity and inclusion. Other domains that had mixed or lower perceived adoption included involving families in organizational feedback and policy making, family peer support, logistical support, and family health and functioning. Adoption of FDC domains differed across staff and organizational characteristics. Conclusions Findings from this mixed methods assessment will inform strategic planning for the scale-up of FDC strategies across JJ agencies in the state, and serve as a template for assessing strengths and weaknesses in the application of family engagement practices in systems nationally

Concentrating pre-mRNA processing factors in the histone locus body facilitates efficient histone mRNA biogenesis

The histone locus body (HLB) assembles at replication-dependent histone genes and concentrates factors required for histone messenger RNA (mRNA) biosynthesis. FLASH (Flice-associated huge protein) and U7 small nuclear RNP (snRNP) are HLB components that participate in 3′ processing of the nonpolyadenylated histone mRNAs by recruiting the endonuclease CPSF-73 to histone pre-mRNA. Using transgenes to complement a FLASH mutant, we show that distinct domains of FLASH involved in U7 snRNP binding, histone pre-mRNA cleavage, and HLB localization are all required for proper FLASH function in vivo. By genetically manipulating HLB composition using mutations in FLASH, mutations in the HLB assembly factor Mxc, or depletion of the variant histone H2aV, we find that failure to concentrate FLASH and/or U7 snRNP in the HLB impairs histone pre-mRNA processing. This failure results in accumulation of small amounts of polyadenylated histone mRNA and nascent read-through transcripts at the histone locus. Thus, the HLB concentrates FLASH and U7 snRNP, promoting efficient histone mRNA biosynthesis and coupling 3′ end processing with transcription termination