The BAF chromatin remodelling complex is an epigenetic regulator of lineage specification in the early mouse embryo.

Dynamic control of gene expression is essential for the development of a totipotent zygote into an embryo with defined cell lineages. The accessibility of genes responsible for cell specification to transcriptional machinery is dependent on chromatin remodelling complexes such as the SWI\SNF (BAF) complex. However, the role of the BAF complex in early mouse development has remained unclear. Here, we demonstrate that BAF155, a major BAF complex subunit, regulates the assembly of the BAF complex in vivo and regulates lineage specification of the mouse blastocyst. We find that associations of BAF155 with other BAF complex subunits become enriched in extra-embryonic lineages just prior to implantation. This enrichment is attributed to decreased mobility of BAF155 in extra-embryonic compared with embryonic lineages. Downregulation of BAF155 leads to increased expression of the pluripotency marker Nanog and its ectopic expression in extra-embryonic lineages, whereas upregulation of BAF155 leads to the upregulation of differentiation markers. Finally, we show that the arginine methyltransferase CARM1 methylates BAF155, which differentially influences assembly of the BAF complex between the lineages and the expression of pluripotency markers. Together, our results indicate a novel role of BAF-dependent chromatin remodelling in mouse development via regulation of lineage specification.

Anteroposterior Limb Skeletal Patterning Requires the Bifunctional Action of SWI/SNF Chromatin Remodeling Complex in Hedgehog Pathway.

Graded Sonic hedgehog (Shh) signaling governs vertebrate limb skeletal patterning along the anteroposterior (AP) axis by regulating the activity of bifunctional Gli transcriptional regulators. The genetic networks involved in this patterning are well defined, however, the epigenetic control of the process by chromatin remodelers remains unknown. Here, we report that the SWI/SNF chromatin remodeling complex is essential for Shh-driven limb AP patterning. Specific inactivation of Srg3/mBaf155, a core subunit of the remodeling complex, in developing limb buds hampered the transcriptional upregulation of Shh/Gli target genes, including the Shh receptor Ptch1 and its downstream effector Gli1 in the posterior limb bud. In addition, Srg3 deficiency induced ectopic activation of the Hedgehog (Hh) pathway in the anterior mesenchyme, resulting in loss of progressive asymmetry. These defects in the Hh pathway accompanied aberrant BMP activity and disruption of chondrogenic differentiation in zeugopod and autopod primordia. Notably, our data revealed that dual control of the Hh pathway by the SWI/SNF complex is essential for spatiotemporal transcriptional regulation of the BMP antagonist Gremlin1, which affects the onset of chondrogenesis. This study uncovers the bifunctional role of the SWI/SNF complex in the Hh pathway to determine the fate of AP skeletal progenitors.

The SWI/SNF chromatin remodeling complex regulates germinal center formation by repressing Blimp-1 expression.

Germinal center (GC) reaction is crucial in adaptive immune responses. The formation of GC is coordinated by the expression of specific genes including Blimp-1 and Bcl-6. Although gene expression is critically influenced by the status of chromatin structure, little is known about the role of chromatin remodeling factors for regulation of GC formation. Here, we show that the SWI/SNF chromatin remodeling complex is required for GC reactions. Mice lacking Srg3/mBaf155, a core component of the SWI/SNF complex, showed impaired differentiation of GC B and follicular helper T cells in response to T cell-dependent antigen challenge. The SWI/SNF complex regulates chromatin structure at the Blimp-1 locus and represses its expression by interacting cooperatively with Bcl-6 and corepressors. The defect in GC reactions in mice lacking Srg3 was due to the derepression of Blimp-1 as supported by genetic studies with Blimp-1-ablated mice. Hence, our study identifies the SWI/SNF complex as a key mediator in GC reactions by modulating Bcl-6-dependent Blimp-1 repression.

The SWI/SNF-like BAF complex is essential for early B cell development.

During the process of B cell development, transcription factors, such as E2A and Ebf1, have been known to play key roles. Although transcription factors and chromatin regulators work in concert to direct the expression of B lineage-specific genes, little is known about the involvement of regulators for chromatin structure during B lymphopoiesis. In this article, we show that deletion of Srg3/mBaf155, a scaffold subunit of the SWI/SNF-like BAF complex, in the hematopoietic lineage caused defects at both the common lymphoid progenitor stage and the transition from pre-pro-B to early pro-B cells due to failures in the expression of B lineage-specific genes, such as Ebf1 and Il7ra, and their downstream target genes. Moreover, mice that were deficient in the expression of Brg1, a subunit of the complex with ATPase activity, also showed defects in early B cell development. We also found that the expression of Ebf1 and Il7ra is directly regulated by the SWI/SNF-like BAF complex. Thus, our results suggest that the SWI/SNF-like BAF complex facilitates early B cell development by regulating the expression of B lineage-specific genes.

The patient-specific mouse model with Foxg1 frameshift mutation uncovers the pathophysiology of FOXG1 syndrome.

Single allelic mutations in the gene encoding the forebrain-specific transcription factor FOXG1 lead to FOXG1 syndrome (FS). Patient-specific animal models are needed to understand the etiology of FS, as FS patients show a wide spectrum of symptoms correlated with location and mutation type in the FOXG1 gene. Here we report the first patient-specific FS mouse model, Q84Pfs heterozygous (Q84Pfs-Het) mice, mimicking one of the most predominant single nucleotide variants in FS. Intriguingly, we found that Q84Pfs-Het mice faithfully recapitulate human FS phenotypes at the cellular, brain structural, and behavioral levels. Importantly, Q84Pfs-Het mice exhibited myelination deficits like FS patients. Further, our transcriptome analysis of Q84Pfs-Het cortex revealed a new role for FOXG1 in synapse and oligodendrocyte development. The dysregulated genes in Q84Pfs-Het brains also predicted motor dysfunction and autism-like phenotypes. Correspondingly, Q84Pfs-Het mice showed movement deficits, repetitive behaviors, increased anxiety, and prolonged behavior arrest. Together, our study revealed the crucial postnatal role of FOXG1 in neuronal maturation and myelination and elucidated the essential pathophysiology mechanisms of FS.

SRG3/mBAF155 stabilizes the SWI/SNF-like BAF complex by blocking CHFR mediated ubiquitination and degradation of its major components.

The murine SWI/SNF-like BAF complex is an ATP-dependent chromatin remodeling complex that functions as a transcriptional regulator in cell proliferation, differentiation and development. The SWI/SNF-like BAF complex consists of several components including core subunits such as BRG1, BAF155/SRG3, BAF47/SNF5/INI1, and BAF170. We have previously shown that the interaction between SRG3/mBAF155 and other components of the complex stabilizes them by attenuating their proteasomal degradation. However, it has not been known how the major components of the SWI/SNF-like BAF complex such as BRG1, SNF5, and BAF60a are targeted for the ubiquitination and degradation, and how SRG3/mBAF155 protects them from the degradation process. Here we report that CHFR interacts with BRG1, SNF5, and BAF60a of the SWI/SNF-like BAF complex and ubiquitinates them to target for degradation through a proteasome-mediated pathway, and that SRG3/mBAF155 stabilizes these components by blocking their interaction with CHFR.

Behavioral Phenotypes of Foxg1 Heterozygous Mice.

FOXG1 syndrome (FS, aka a congenital variant of Rett syndrome) is a recently defined rare and devastating neurodevelopmental disorder characterized by various symptoms, including severe intellectual disability, autistic features, involuntary, and continuous jerky movements, feeding problems, sleep disturbances, seizures, irritability, and excessive crying. FS results from mutations in a single allele of the FOXG1 gene, leading to impaired FOXG1 function. Therefore, in establishing mouse models for FS, it is important to test if heterozygous (HET) mutation in the Foxg1 gene, mimicking genotypes of the human FS individuals, also manifests phenotypes similar to their symptoms. We analyzed HET mice with a null mutation allele in a single copy of Foxg1, and found that they show various phenotypes resembling the symptoms of the human FS individuals. These include increased anxiety in the open field as well as impairment in object recognition, motor coordination, and fear learning and contextual and cued fear memory. Our results suggest that Foxg1 HET mice recapitulate at least some symptoms of the human FS individuals.

BAP1 shapes the bone marrow niche for lymphopoiesis by fine-tuning epigenetic profiles in endosteal mesenchymal stromal cells.

Hematopoiesis occurs within a unique bone marrow (BM) microenvironment, which consists of various niche cells, cytokines, growth factors, and extracellular matrix components. These multiple components directly or indirectly regulate the maintenance and differentiation of hematopoietic stem cells (HSCs). Here we report that BAP1 in BM mesenchymal stromal cells (MSCs) is critical for the maintenance of HSCs and B lymphopoiesis. Mice lacking BAP1 in MSCs show aberrant differentiation of hematopoietic stem and progenitor cells, impaired B lymphoid differentiation, and expansion of myeloid lineages. Mechanistically, BAP1 loss in distinct endosteal MSCs, expressing PRX1 but not LEPR, leads to aberrant expression of genes affiliated with BM niche functions. BAP1 deficiency leads to a reduced expression of pro-hematopoietic factors such as Scf caused by increased H2AK119-ub1 and H3K27-me3 levels on the promoter region of these genes. On the other hand, the expression of myelopoiesis stimulating factors including Csf3 was increased by enriched H3K4-me3 and H3K27-ac levels on their promoter, causing myeloid skewing. Notably, loss of BAP1 substantially blocks B lymphopoiesis and skews the differentiation of hematopoietic precursors toward myeloid lineages in vitro, which is reversed by G-CSF neutralization. Thus, our study uncovers a key role for BAP1 expressed in endosteal MSCs in controlling normal hematopoiesis in mice by modulating expression of various niche factors governing lymphopoiesis and myelopoiesis via histone modifications.

Bap1/SMN axis in Dpp4+ skeletal muscle mesenchymal cells regulates the neuromuscular system.

The survival of motor neuron (SMN) protein is a major component of the pre-mRNA splicing machinery and is required for RNA metabolism. Although SMN has been considered a fundamental gene for the central nervous system, due to its relationship with neuromuscular diseases, such as spinal muscular atrophy, recent studies have also revealed the requirement of SMN in non-neuronal cells in the peripheral regions. Here, we report that the fibro-adipogenic progenitor subpopulation expressing Dpp4 (Dpp4+ FAPs) is required for the neuromuscular system. Furthermore, we also reveal that BRCA1-associated protein-1 (Bap1) is crucial for the stabilization of SMN in FAPs by preventing its ubiquitination-dependent degradation. Inactivation of Bap1 in FAPs decreased SMN levels and accompanied degeneration of the neuromuscular junction, leading to loss of motor neurons and muscle atrophy. Overexpression of the ubiquitination-resistant SMN variant, SMNK186R, in Bap1-null FAPs completely prevented neuromuscular degeneration. In addition, transplantation of Dpp4+ FAPs, but not Dpp4- FAPs, completely rescued neuromuscular defects. Our data reveal the crucial role of Bap1-mediated SMN stabilization in Dpp4+ FAPs for the neuromuscular system and provide the possibility of cell-based therapeutics to treat neuromuscular diseases.

The histone H3-lysine 4-methyltransferase Mll4 regulates the development of growth hormone-releasing hormone-producing neurons in the mouse hypothalamus.

In humans, inactivating mutations in MLL4, which encodes a histone H3-lysine 4-methyltransferase, lead to Kabuki syndrome (KS). While dwarfism is a cardinal feature of KS, the underlying etiology remains unclear. Here we report that Mll4 regulates the development of growth hormone-releasing hormone (GHRH)-producing neurons in the mouse hypothalamus. Our two Mll4 mutant mouse models exhibit dwarfism phenotype and impairment of the developmental programs for GHRH-neurons. Our ChIP-seq analysis reveals that, in the developing mouse hypothalamus, Mll4 interacts with the transcription factor Nrf1 to trigger the expression of GHRH-neuronal genes. Interestingly, the deficiency of Mll4 results in a marked reduction of histone marks of active transcription, while treatment with the histone deacetylase inhibitor AR-42 rescues the histone mark signature and restores GHRH-neuronal production in Mll4 mutant mice. Our results suggest that the developmental dysregulation of Mll4-directed epigenetic control of transcription plays a role in the development of GHRH-neurons and dwarfism phenotype in mice.

Twist2 promotes CD8+ T-cell differentiation by repressing ThPOK expression.

CD4/CD8 T-cell lineage differentiation is a key process in immune system development; however, a defined regulator(s) that converts the signal from T-cell receptor and co-receptor complexes into lineage differentiation remains unclear. Here, we show that Twist2 is a critical factor in CD4/CD8 thymocyte differentiation. Twist2 expression is differentially regulated by T-cell receptor signaling, leading to differentiation into the CD4 or CD8 lineage. Forced Twist2 expression perturbed CD4+ thymocyte differentiation while enhancing CD8+ thymocyte differentiation. Furthermore, Twist2 expression produced mature CD8+ thymocytes in B2m-/- mice, while its deficiency significantly impaired CD8+ cells in MHC class-II-/- and TCR transgenic mice, favoring CD8 T-cell differentiation. During CD8 lineage differentiation, Twist2 interacted with Runx3 to bind to the silencer region of the ThPOK locus, thereby blocking ThPOK expression. These findings indicate that Twist2 is a part of the transcription factor network controlling CD8 lineage differentiation.

SRG3, a core component of mouse SWI/SNF complex, is essential for extra-embryonic vascular development.

The SWI/SNF chromatin-remodeling complex functions as a transcriptional regulator and plays a significant role in cell proliferation, differentiation and embryonic development. SRG3, a homologue of human BAF155, is a core component of the mouse SWI/SNF chromatin remodeling complex. Mutant mice deficient in Srg3 expression are peri-implantation lethal. To investigate the role of SRG3 in the post-implantation stage, we generated SRG3 transgene-rescued (Srg3-/-Tg+) embryos by inducing exogenous gene expression. These Srg3-/-Tg+ embryos overcame early embryonic lethality and extended the life span until mid-gestation. However, the embryos displayed significant defects in blood vessel formation and fetal circulation within the yolk sac around embryonic day 10.5, which led to developmental retardation and death. We found that SRG3 expression was absent in the visceral endoderm of Srg3-/-Tg+ yolk sacs, while SRG3 was normally expressed in wild-type embryos. In addition, expression of angiogenesis-related genes, including Angiopoietin1, Tie2, EphrinB2, Ihh and Notch1, was markedly reduced in Srg3-/-Tg+ yolk sacs. During normal angiogenesis, maturation of the visceral endoderm development is observed in the yolk sac. However, in Srg3-/-Tg+ yolk sacs, the visceral endoderm did not develop normally. Our results indicate that SRG3 is required for angiogenesis and visceral endoderm development in the yolk sac.

Critical roles of ARHGAP36 as a signal transduction mediator of Shh pathway in lateral motor columnar specification.

During spinal cord development, Sonic hedgehog (Shh), secreted from the floor plate, plays an important role in the production of motor neurons by patterning the ventral neural tube, which establishes MN progenitor identity. It remains unknown, however, if Shh signaling plays a role in generating columnar diversity of MNs that connect distinct target muscles. Here, we report that Shh, expressed in MNs, is essential for the formation of lateral motor column (LMC) neurons in vertebrate spinal cord. This novel activity of Shh is mediated by its downstream effector ARHGAP36, whose expression is directly induced by the MN-specific transcription factor complex Isl1-Lhx3. Furthermore, we found that AKT stimulates the Shh activity to induce LMC MNs through the stabilization of ARHGAP36 proteins. Taken together, our data reveal that Shh, secreted from MNs, plays a crucial role in generating MN diversity via a regulatory axis of Shh-AKT-ARHGAP36 in the developing mouse spinal cord.

Prescribing Patterns of Antihypertensives for Treatment-Naïve Patients in South Korea: From Korean NHISS Claim Data.

BACKGROUND: Several factors influence the choice of antihypertensive drugs. To facilitate the rational use of drugs it is important to assess their prescription patterns over time. This study aims to evaluate doctors' prescribing patterns of antihypertensive drugs for drug-naïve patients in South Korea. METHODS: The claims data of the Korean National Health Insurance Research Database from 1 January 2011 to 31 December 2015 were analyzed. The data virtually cover the entire South Korean population. Antihypertensive drugs were further subdivided into angiotensin receptor blockers (ARBs), angiotensin-converting enzyme inhibitors (ACEis), calcium channel blockers (CCBs), beta-blockers (BBs), and thiazide diuretics. The prescription pattern of antihypertensive drugs and associated factors were assessed according to the patients' characteristics, including associated comorbidities. RESULTS: A total of 2,919,162 subjects had started taking antihypertension medications during the study period. ARB was the most frequently prescribed drug (51.6%) followed by CCB (45.0%), BB (18.5%), diuretics (17.0%), and ACEi (11.7%). Most patients were prescribed with monotherapy (66.7%) rather than combination therapy (33.3%), and CCB was the most frequently prescribed monotherapy drug (25.7%). For combination therapy, ARB + CCB was the most frequently prescribed combination, and the prescription frequency was found to be increasing. In patients prescribed with combination therapy, most had been prescribed single-pill fixed-dose combination. CONCLUSION: We identified the physicians' prescription patterns of antihypertensive drugs for treatment-naïve patients. The findings of this study can lead to a rational, evidence-based, and cost-effective improvement of prescription patterns in newly diagnosed hypertensive patients.

Marital status integration and suicide: A meta-analysis and meta-regression.

Marital status is an index of the phenomenon of social integration within social structures and has long been identified as an important predictor suicide. However, previous meta-analyses have focused only on a particular marital status, or not sufficiently explored moderators. A meta-analysis of observational studies was conducted to explore the relationships between marital status and suicide and to understand the important moderating factors in this association. Electronic databases were searched to identify studies conducted between January 1, 2000 and June 30, 2016. We performed a meta-analysis, subgroup analysis, and meta-regression of 170 suicide risk estimates from 36 publications. Using random effects model with adjustment for covariates, the study found that the suicide risk for non-married versus married was OR = 1.92 (95% CI: 1.75-2.12). The suicide risk was higher for non-married individuals aged <65 years than for those aged ≥65 years, and higher for men than for women. According to the results of stratified analysis by gender, non-married men exhibited a greater risk of suicide than their married counterparts in all sub-analyses, but women aged 65 years or older showed no significant association between marital status and suicide. The suicide risk in divorced individuals was higher than for non-married individuals in both men and women. The meta-regression showed that gender, age, and sample size affected between-study variation. The results of the study indicated that non-married individuals have an aggregate higher suicide risk than married ones. In addition, gender and age were confirmed as important moderating factors in the relationship between marital status and suicide.