Understanding Multiple Independent Functions of the Tip60 Acetyltransferase in Embryonic Development

Chromatin is a dynamic structure, and chromatin remodeling enzymes regulate chromatin structure to control gene expression and proper lineage specification. Tip60-p400 is a multi-subunit chromatin remodeling complex containing two biochemical activities: the Tip60 subunit is a lysine acetyltransferase (KAT) that targets histones and non-histone proteins, and p400 catalyzes ATP-dependent incorporation of histone variant H2AZ into chromatin. Both of these chromatin modifications have been widely studied with respect to gene expression, DNA damage repair, and apoptosis. Ablation of these catalytic subunits causes defects in normal embryonic development, ESC self-renewal, and gene expression. My goal has been to understand the multiple independent functions of Tip60-p400 acetyltransferase in ESC maintenance and embryonic development. I showed that Tip60 KAT function is dispensable for gene expression, chromatin accessibility, and ESC self-renewal, which is different from Tip60 knockdown phenotype. Interestingly, KAT deficient mutants exhibited defect in differentiation towards mesoderm and endoderm lineages. Consistent with this defect, I also observed gastrulation defect in mice lacking Tip60 KAT activity. Together, these data demonstrate that Tip60 KAT dependent function is only required during later stages of embryonic development, and it is dispensable for ESC self-renewal and pre-implantation development. Tip60 KAT contains four isoforms generated from alternative splicing, whose individual functions are poorly characterized. In the second part of this thesis, I investigated the developmental role of one of the isoforms of Tip60, called Tip55. Unlike Tip60 knockout mice, which lack all the isoforms and causes pre-implantation lethality, I found that ablation of Tip55 results in post-implantation lethality. I further found that loss of Tip55 causes defects in heart, and neural tube development, demonstrating the essential function of Tip55 isoform for organogenesis during embryonic development. Together, these studies have provided new insight into the functions of Tip60-p400 and the mechanisms by which this complex regulates gene expression, ESC pluripotency, and embryonic development. Furthermore, these studies set the stage for future work to identify how the catalytic and non-catalytic functions are directed to perform distinct regulatory functions, as well as how each Tip60 isoform individually contributes to formation of the mammalian body plan

KAT-Independent Gene Regulation by Tip60 Promotes ESC Self-Renewal but Not Pluripotency

Although histone-modifying enzymes are generally assumed to function in a manner dependent on their enzymatic activities, this assumption remains untested for many factors. Here, we show that the Tip60 (Kat5) lysine acetyltransferase (KAT), which is essential for embryonic stem cell (ESC) self-renewal and pre-implantation development, performs these functions independently of its KAT activity. Unlike ESCs depleted of Tip60, KAT-deficient ESCs exhibited minimal alterations in gene expression, chromatin accessibility at Tip60 binding sites, and self-renewal, thus demonstrating a critical KAT-independent role of Tip60 in ESC maintenance. In contrast, KAT-deficient ESCs exhibited impaired differentiation into mesoderm and endoderm, demonstrating a KAT-dependent function in differentiation. Consistent with this phenotype, KAT-deficient mouse embryos exhibited post-implantation developmental defects. These findings establish separable KAT-dependent and KAT-independent functions of Tip60 in ESCs and during differentiation, revealing a complex repertoire of regulatory functions for this essential chromatin remodeling complex

TIP55, a splice isoform of the KAT5 acetyltransferase, is essential for developmental gene regulation and organogenesis

Regulation of chromatin structure is critical for cell type-specific gene expression. Many chromatin regulatory complexes exist in several different forms, due to alternative splicing and differential incorporation of accessory subunits. However, in vivo studies often utilize mutations that eliminate multiple forms of complexes, preventing assessment of the specific roles of each. Here we examined the developmental roles of the TIP55 isoform of the KAT5 histone acetyltransferase. In contrast to the pre-implantation lethal phenotype of mice lacking all four Kat5 transcripts, mice specifically deficient for Tip55 die around embryonic day 11.5 (E11.5). Prior to developmental arrest, defects in heart and neural tube were evident in Tip55 mutant embryos. Specification of cardiac and neural cell fates appeared normal in Tip55 mutants. However, cell division and survival were impaired in heart and neural tube, respectively, revealing a role for TIP55 in cellular proliferation. Consistent with these findings, transcriptome profiling revealed perturbations in genes that function in multiple cell types and developmental pathways. These findings show that Tip55 is dispensable for the pre- and early post-implantation roles of Kat5, but is essential during organogenesis. Our results raise the possibility that isoform-specific functions of other chromatin regulatory proteins may play important roles in development

Factors associated with nutritional status of women of reproductive age group in rural, Nepal

Background: Maternal nutrition is one of the most important health and welfare problems among women in developing countries.In women of reproductive age, malnutrition can result in adverse pregnancy outcomes. Maternal nutrition is the major publichealth problem in Nepal. Objective: The main purpose of the study was to establish the factors influencing the nutritional statusof non-pregnant and non-lactating women of reproductive age in Shree Kedar VDC, Baitadi of Nepal. Methodology: A descriptive,cross-sectional study involving 229 non-pregnant and non-lactating women of reproductive age (15-49) was carried out in Baitadidistrict of Nepal. Results: In a total of 229 women, 45.4% of women were of age group 20-29 years. A significant proportion 32.3%was underweight with a mean body mass index of <18.5% and 4.8% were either overweight or obese. Women of all age groups werevulnerable to undernutrition. There was an indication of insufficient food availability at the household level. 22.7% of the studypopulation reported of food inadequacy. Educational status and marital status were statistically significant in the determination ofnutritional status. The study showed that the nutrition status of the study population in Shree Kedar VDC was poor. Conclusion:Food inadequacy, inadequate information/knowledge, low educational levels, caste, income, and family size were the key contributorsto poor nutritional status. Furthermore, nutritional interventions are highly needed to improve the nutrition status of women

R loops regulate promoter-proximal chromatin architecture and cellular differentiation

Numerous chromatin-remodeling factors are regulated by interactions with RNA, although the contexts and functions of RNA binding are poorly understood. Here we show that R loops, RNA-DNA hybrids consisting of nascent transcripts hybridized to template DNA, modulate the binding of two key chromatin-regulatory complexes, Tip60-p400 and polycomb repressive complex 2 (PRC2) in mouse embryonic stem cells (ESCs). Like PRC2, the Tip60-p400 histone acetyltransferase complex binds to nascent transcripts; however, transcription promotes chromatin binding of Tip60-p400 but not PRC2. Interestingly, we observed higher Tip60-p400 and lower PRC2 levels at genes marked by promoter-proximal R loops. Furthermore, disruption of R loops broadly decreased Tip60-p400 occupancy and increased PRC2 occupancy genome wide. In agreement with these alterations, ESCs partially depleted of R loops exhibited impaired differentiation. These results show that R loops act both positively and negatively in modulating the recruitment of key pluripotency regulators

Developing and deploying a community healthcare worker-driven, digitally- enabled integrated care system for municipalities in rural Nepal

International audienceIntegrating care at the home and facility level is a critical yet neglected function of healthcare delivery systems. There are few examples in practice or in the academic literature of affordable, digitally-enabled integrated care approaches embedded within healthcare delivery systems in low- and middle-income countries. Simultaneous advances in affordable digital technologies and community healthcare workers offer an opportunity to address this challenge. We describe the development of an integrated care system involving community healthcare worker networks that utilize a home-to-facility electronic health record platform for rural municipalities in Nepal. Key aspects of our approach of relevance to a global audience include: community healthcare workers continuously engaging with populations through household visits every three months; community healthcare workers using digital tools during the routine course of clinical care; individual and population-level data generated routinely being utilized for program improvement; and being responsive to privacy, security, and human rights concerns. We discuss implementation, lessons learned, challenges, and opportunities for future directions in integrated care delivery systems