Full methylation of H3K27 by PRC2 is dispensable for initial embryoid body formation but required to maintain differentiated cell identity.

Polycomb repressive complex 2 (PRC2) catalyzes methylation of histone H3 on lysine 27 and is required for normal development of complex eukaryotes. The nature of that requirement is not clear. H3K27me3 is associated with repressed genes, but the modification is not sufficient to induce repression and, in some instances, is not required. We blocked full methylation of H3K27 with both a small molecule inhibitor, GSK343, and by introducing a point mutation into EZH2, the catalytic subunit of PRC2, in the mouse CJ7 cell line. Cells with substantively decreased H3K27 methylation differentiate into embryoid bodies, which contrasts with EZH2 null cells. PRC2 targets had varied requirements for H3K27me3, with a subset that maintained normal levels of repression in the absence of methylation. The primary cellular phenotype of blocked H3K27 methylation was an inability of altered cells to maintain a differentiated state when challenged. This phenotype was determined by H3K27 methylation in embryonic stem cells through the first 4 days of differentiation. Full H3K27 methylation therefore was not necessary for formation of differentiated cell states during embryoid body formation but was required to maintain a stable differentiated state.

BRIDGE to liver health: implementation of a group telehealth psychoeducational program through shared medical appointments for MASLD management.

BACKGROUND: Metabolic dysfunction-associated steatotic liver disease (MASLD) represents a significantly costly and increasingly prevalent disease, with treatment focused on lifestyle intervention. Integrating education and behavioral health into clinical care offers opportunities to engage and empower patients to prevent progression of liver disease. We describe the design and implementation of Behavioral Resources and Intervention through Digital Group Education (BRIDGE), a 6-session group telehealth program led by advanced practice providers (APPs) in 90-min shared medical appointments (SMAs) with small groups of MASLD patients in an academic outpatient hepatology clinic. The program contains multi-component group interventions, with didactic education and behavioral coaching, while leveraging peer-based learning and support. METHODS: A mixed-methods exploratory pilot study was conducted. Feasibility and acceptability of the clinical intervention were assessed by tracking recruitment, attendance, and retention of BRIDGE participants, patient interviews, and debriefing of clinician and staff views of the clinical program. Implementation metrics included program development time, workflow and scheduling logistics, and billing compliance for sustainability. Finally, patient parameters including changes in liver enzymes, FIB-4, weight, and BMI from pre- to post-BRIDGE were retrospectively analyzed. RESULTS: We included 57 participants (median age 57, interquartile range (IQR) 50 - 65 years), 38 (67%) female, 38 (67%) white, and 40% had public insurance. Thirty-three (58%) participants completed all six sessions, while 43 (75%) attended at least five sessions. Patients who completed all sessions were older (median age 61 vs 53.5; p = 0.01). Gender, race/ethnicity, and insurance type were not significantly associated with missed sessions, and patients had similar rates of completion regardless of weight, BMI, or stage of liver disease. Barriers to completion included personal illness, family reasons, work commitments, or insurance issues. Prior to BRIDGE, median BMI was 31.9 (SD 29 - 36), with a median weight loss of 2 pounds (IQR -2 - 6) after BRIDGE. CONCLUSION: The BRIDGE telehealth SMA program was feasible, well-attended, and positively reviewed. This pilot study informs future iterations of program development and evaluation of outcome measures.

The effect of the food environment on fresh produce served in family child care homes.

BACKGROUND: Higher produce consumption in childhood decreases risks of short- and long-term malnutrition, obesity, and disease. Children in early care programs, including family child care homes (FCCHs), receive 50-67% of daily nutrition while in care. Procuring nutritious foods requires grocer access, which is absent in food deserts (FDs). AIM: To determine if FCCH food environment (FE) impacted distance to grocers and amount of fresh produce served. METHODS: Using a cross-sectional design, Modified Retail Food Environment Index scores determined census tract FD status. FCCH and grocer addresses were geocoded and distance to the nearest grocers was calculated. Fresh produce was observed during two lunches. RESULTS: FE did not influence distance to grocers or fresh produce served. Non-desert FCCHs tended to serve fresh produce more frequently. The amount of fresh produce served was overall low. CONCLUSION: Further studies are warranted to inform policies aimed to reduce provider barriers regarding service of fresh produce.

CCCTC-binding factor and the transcription factor T-bet orchestrate T helper 1 cell-specific structure and function at the interferon-gamma locus.

How cell type-specific differences in chromatin conformation are achieved and their contribution to gene expression are incompletely understood. Here we identify a cryptic upstream orchestrator of interferon-gamma (IFNG) transcription, which is embedded within the human IL26 gene, compromised of a single CCCTC-binding factor (CTCF) binding site and retained in all mammals, even surviving near-complete evolutionary deletion of the equivalent gene encoding IL-26 in rodents. CTCF and cohesins occupy this element in vivo in a cell type-nonspecific manner. This element is juxtaposed to two other sites located within the first intron and downstream of Ifng, where CTCF, cohesins, and the transcription factor T-bet bind in a T helper 1 (Th1) cell-specific manner. These interactions, close proximity of other elements within the locus to each other and to the gene encoding interferon-gamma, and robust murine Ifng expression are dependent on CTCF and T-bet. The results demonstrate that cooperation between architectural (CTCF) and transcriptional enhancing (T-bet) factors and the elements to which they bind is required for proper Th1 cell-specific expression of Ifng.

Jmjd3 and UTX play a demethylase-independent role in chromatin remodeling to regulate T-box family member-dependent gene expression.

The stable and heritable H3K27-methyl mark suppresses transcription of lineage-specific genes in progenitor cells. During developmental transitions, histone demethylases are required to dramatically alter epigenetic and gene expression states to create new cell-specific profiles. It is unclear why demethylase proteins that antagonize polycomb-mediated repression continue to be expressed in terminally differentiated cells where further changes in H3K27 methylation could be deleterious. In this study, we show that the H3K27 demethylases, Jmjd3 and UTX, mediate a functional interaction between the lineage-defining T-box transcription factor family and a Brg1-containing SWI/SNF remodeling complex. Importantly, Jmjd3 is required for the coprecipitation of Brg1 with the T-box factor, T-bet, and this interaction is necessary for Ifng remodeling in differentiated Th1 cells. Thus, Jmjd3 has a required role in general chromatin remodeling that is independent from its H3K27 demethylase potential. This function for H3K27 demethylase proteins may explain their presence in differentiated cells where the epigenetic profile is already established.

Molecular mechanisms by which T-bet regulates T-helper cell commitment.

Current research suggests that a number of newly identified T-helper cell subsets retain a degree of context-dependent plasticity in their signature cytokine expression patterns. To understand this process, a major challenge is to determine the molecular mechanisms by which lineage-defining transcription factors regulate gene expression profiles in T-helper cells. This mechanistic information will aid in our interpretation of whether a T-helper cell state that expresses or retains the capacity to re-express a combination of lineage-defining transcription factors will have a stable or more flexible gene expression profile. Studies examining the developmental T-box transcription factor T-bet demonstrate the powerful information that is gained from combining in vivo analysis with basic biochemical and molecular mechanism approaches. Significantly, T-bet's ability to physically recruit epigenetic modifying complexes, in particular a Jmjd3 H3K27-demethylase and a Set7/9 H3K4-methyltransferase complex, to its target genes allows T-bet to effectively reverse and establish new epigenetic states. This observation suggests that until T-bet is permanently extinguished, T-helper cells will retain some plasticity toward a T-helper 1-like program. Therefore, insight into the complexity of T-helper cell commitment decisions will be aided by determining the molecular mechanisms for lineage-defining transcription factors.

Suicide, guns, and public policy.

Suicide is a serious public health concern that is responsible for almost 1 million deaths each year worldwide. It is commonly an impulsive act by a vulnerable individual. The impulsivity of suicide provides opportunities to reduce the risk of suicide by restricting access to lethal means. In the United States, firearms, particularly handguns, are the most common means of suicide. Despite strong empirical evidence that restriction of access to firearms reduces suicides, access to firearms in the United States is generally subject to few restrictions. Implementation and evaluation of measures such as waiting periods and permit requirements that restrict access to handguns should be a top priority for reducing deaths from impulsive suicide in the United States.

Restricted MHC-peptide repertoire predisposes to autoimmunity.

MHC molecules associated with autoimmunity possess known structural features that limit the repertoire of peptides that they can present. Such limitation gives a selective advantage to TCRs that rely on interaction with the MHC itself, rather than with the peptide residues. At the same time, negative selection is impaired because of the lack of negatively selecting peptide ligands. The combination of these factors may predispose to autoimmunity. We found that mice with an MHC class II-peptide repertoire reduced to a single complex demonstrated various autoimmune reactions. Transgenic mice bearing a TCR (MM14.4) cloned from such a mouse developed severe autoimmune dermatitis. Although MM14.4 originated from a CD4+ T cell, dermatitis was mediated by CD8+ T cells. It was established that MM14.4+ is a highly promiscuous TCR with dual MHC class I/MHC class II restriction. Furthermore, mice with a limited MHC-peptide repertoire selected elevated numbers of TCRs with dual MHC class I/MHC class II restriction, a likely source of autoreactivity. Our findings may help to explain the link between MHC class I responses that are involved in major autoimmune diseases and the well-established genetic linkage of these diseases with MHC class II.

Cell type-specific induction and inhibition of apoptosis by Herpes Simplex virus type 2 ICP10.

Herpes simplex virus (HSV) inhibits apoptosis induced by external stimuli in epithelial cells. In contrast, apoptosis is the primary outcome in HSV-infected lymphocytes. Here, we show that HSV type 2 (HSV-2) gene expression appears to be necessary for the induction of apoptosis in Jurkat cells, a T-cell leukemia line. HSV-2 ICP10 gene expression is sufficient to induce apoptosis in Jurkat cells, while its expression protects epithelial HEp-2 cells from apoptosis triggered by cycloheximide and tumor necrosis factor alpha. Thus, the effect of HSV-2 gene expression on the cellular apoptotic pathway appears to depend on the specific cell type.

Common themes emerge in the transcriptional control of T helper and developmental cell fate decisions regulated by the T-box, GATA and ROR families.

Cellular differentiation requires the precise action of lineage-determining transcription factors. In the immune system, CD4(+) T helper cells differentiate into at least three distinct effector lineages, T helper type 1 (Th1), Th2 and Th17, with the fate of the cell at least in part determined by the transcription factors T-box expressed in T cells (T-bet), GATA-3 and retinoid-related orphan receptor gammat (RORgammat), respectively. Importantly, these transcription factors are members of larger families that are required for numerous developmental transitions from early embryogenesis into adulthood. Mutations in members of these transcription factor families are associated with a number of human genetic diseases due to a failure in completing lineage-specification events when the factor is dysregulated. Mechanistically, there are both common and distinct functional activities that are utilized by T-box, GATA and ROR family members to globally alter the cellular gene expression profiles at specific cell fate decision checkpoints. Therefore, understanding the molecular events that contribute to the ability of T-bet, GATA-3 and RORgammat to define T helper cell lineages can provide valuable information relevant to the establishment of other developmental systems and, conversely, information from diverse developmental systems may provide unexpected insights into the molecular mechanisms utilized in T helper cell differentiation.

Ataxin-7 and Non-stop coordinate SCAR protein levels, subcellular localization, and actin cytoskeleton organization.

Atxn7, a subunit of SAGA chromatin remodeling complex, is subject to polyglutamine expansion at the amino terminus, causing spinocerebellar ataxia type 7 (SCA7), a progressive retinal and neurodegenerative disease. Within SAGA, the Atxn7 amino terminus anchors Non-stop, a deubiquitinase, to the complex. To understand the scope of Atxn7-dependent regulation of Non-stop, substrates of the deubiquitinase were sought. This revealed Non-stop, dissociated from Atxn7, interacts with Arp2/3 and WAVE regulatory complexes (WRC), which control actin cytoskeleton assembly. There, Non-stop countered polyubiquitination and proteasomal degradation of WRC subunit SCAR. Dependent on conserved WRC interacting receptor sequences (WIRS), Non-stop augmentation increased protein levels, and directed subcellular localization, of SCAR, decreasing cell area and number of protrusions. In vivo, heterozygous mutation of SCAR did not significantly rescue knockdown of Atxn7, but heterozygous mutation of Atxn7 rescued haploinsufficiency of SCAR.

An essential interaction between T-box proteins and histone-modifying enzymes.

Cellular differentiation requires precisely coordinated events to induce developmentally appropriate gene expression profiles. Lineage-defining transcription factors are responsible for establishing cell-type specific gene expression patterns during development. Recently, we reported a novel mechanism by which the T-box transcription factor T-bet interacts with JMJD3, an H3K27-demethylase, and Set7/9, an H3K4-methyltransferase (Genes Dev. 2008. 22: 2980-2993). Importantly, separable contact points in the T-box DNA binding domain mediate these interactions. Due to the highly conserved nature of the contact residues, these represent common interactions for the T-box family. Therefore, studies examining the molecular mechanisms that account for the ability of T-bet to regulate Ifng and Cxcr3, prototypic CD4+ Th1 genes, have provided novel insight into essential regulatory events that occur at diverse developmental transitions. In this article, we discuss the implications for these findings as well as explore the role epigenetic mechanisms may play in the development of human genetic diseases that are caused by T-box mutations, including congenital heart defects, cleft palate, pituitary deficiencies, and Ulnar-mammary syndrome.

T-bet-dependent S1P5 expression in NK cells promotes egress from lymph nodes and bone marrow.

During a screen for ethylnitrosourea-induced mutations in mice affecting blood natural killer (NK) cells, we identified a strain, designated Duane, in which NK cells were reduced in blood and spleen but increased in lymph nodes (LNs) and bone marrow (BM). The accumulation of NK cells in LNs reflected a decreased ability to exit into lymph. This strain carries a point mutation within Tbx21 (T-bet), which generates a defective protein. Duane NK cells have a 30-fold deficiency in sphingosine-1-phosphate receptor 5 (S1P5) transcript levels, and S1P5-deficient mice exhibit an egress defect similar to Duane. Chromatin immunoprecipitation confirms binding of T-bet to the S1pr5 locus. S1P-deficient mice exhibit a more severe NK cell egress block, and the FTY720-sensitive S1P1 also plays a role in NK cell egress from LNs. S1P5 is not inhibited by CD69, a property that may facilitate trafficking of activated NK cells to effector sites. Finally, the accumulation of NK cells within BM of S1P-deficient mice was associated with reduced numbers in BM sinusoids, suggesting a role for S1P in BM egress. In summary, these findings identify S1P5 as a T-bet-induced gene that is required for NK cell egress from LNs and BM.

Coordinated but physically separable interaction with H3K27-demethylase and H3K4-methyltransferase activities are required for T-box protein-mediated activation of developmental gene expression.

During cellular differentiation, both permissive and repressive epigenetic modifications must be negotiated to create cell-type-specific gene expression patterns. The T-box transcription factor family is important in numerous developmental systems ranging from embryogenesis to the differentiation of adult tissues. By analyzing point mutations in conserved sequences in the T-box DNA-binding domain, we found that two overlapping, but physically separable regions are required for the physical and functional interaction with H3K27-demethylase and H3K4-methyltransferase activities. Importantly, the ability to associate with these histone-modifying complexes is a conserved function for the T-box family. These novel mechanisms for T-box-mediated epigenetic regulation are essential, because point mutations that disrupt these interactions are found in a diverse array of human developmental genetic diseases.

T-bet's ability to regulate individual target genes requires the conserved T-box domain to recruit histone methyltransferase activity and a separate family member-specific transactivation domain.

Appropriate cellular differentiation and specification rely upon the ability of key developmental transcription factors to precisely establish gene expression patterns. These transcription factors often regulate epigenetic events. However, it has been unclear whether this is the only role that they play in functionally regulating developmental gene expression pathways or whether they also participate in downstream transactivation events at the same promoter. The T-box transcription factor family is important in cellular specification events in many developmental systems, and determining the molecular mechanisms by which this family regulates gene expression networks warrants attention. Here, we examine the mechanism by which T-bet, a critical T-box protein in the immune system, influences transcription. T-bet is both necessary and sufficient to induce permissive histone H3-K4 dimethyl modifications at the CXCR3 and IFN-gamma promoters. A T-bet structure-function analysis revealed that the conserved T-box domain, with a small C-terminal portion, is required for recruiting histone methyltransferase activity to promoters. Interestingly, this function is conserved in the T-box family and is necessary, but not sufficient, to induce transcription, with an independent transactivation activity also required. The requirement for two separable functional activities may ultimately contribute to the stringent role for T-box proteins in establishing specific developmental gene expression pathways.

Apoptosis and antigen receptor function in T and B cells following exposure to herpes simplex virus.

T cells are an essential component of the immune response against herpes simplex virus (HSV) infection. We previously reported that incubation of T cells with HSV-infected fibroblasts inhibits subsequent T cell antigen receptor signal transduction. In the current study, we found that incubation of T cells with HSV-infected fibroblasts also leads to apoptosis in exposed T cells. Apoptosis was observed in Jurkat cells, a T cell leukemia line, and also in CD4(+) cells isolated from human peripheral blood mononuclear cells. Direct infection of these cells with HSV also resulted in apoptosis. Clinical isolates of both HSV type 1 and 2 induced apoptosis in infected T cells at comparable levels to cells infected with laboratory strains of HSV, suggesting an immune evasion mechanism that may be clinically relevant. Further understanding of these viral immune evasion mechanisms could be exploited for better management of HSV infection.

Isolation and structure determination of an antimicrobial ester from a marine sediment-derived bacterium.

A new compound, assigned the trivial name bonactin (1), has been isolated from the liquid culture of a Streptomyces sp. BD21-2 obtained from a shallow-water sediment sample collected at Kailua Beach, Oahu, Hawaii. Structure elucidation employed one- and two-dimensional NMR, HRFABMS, IR, and chemical analysis. Bonactin displayed antimicrobial activity against both Gram-positive and Gram-negative bacteria as well as antifungal activity.