Lsh/HELLS is required for B lymphocyte development and immunoglobulin class switch recombination.

Mutation of HELLS (Helicase, Lymphoid-Specific)/Lsh in human DNA causes a severe immunodeficiency syndrome, but the nature of the defect remains unknown. We assessed here the role of Lsh in hematopoiesis using conditional Lsh knockout mice with expression of Mx1 or Vav Cre-recombinase. Bone marrow transplantation studies revealed that Lsh depletion in hematopoietic stem cells severely reduced B cell numbers and impaired B cell development in a hematopoietic cell-autonomous manner. Lsh-deficient mice without bone marrow transplantation exhibited lower Ig levels in vivo compared to controls despite normal peripheral B cell numbers. Purified B lymphocytes proliferated normally but produced less immunoglobulins in response to in vitro stimulation, indicating a reduced capacity to undergo class switch recombination (CSR). Analysis of germline transcripts, examination of double-stranded breaks using biotin-labeling DNA break assay, and End-seq analysis indicated that the initiation of the recombination process was unscathed. In contrast, digestion-circularization PCR analysis and high-throughput sequencing analyses of CSR junctions and a chromosomal break repair assay indicated an impaired ability of the canonical end-joining pathway in Lsh-deficient B cells. Our data suggest a hematopoietic cell-intrinsic role of Lsh in B cell development and in CSR providing a potential target for immunodeficiency therapy.

Genome-wide DNA methylation patterns in LSH mutant reveals de-repression of repeat elements and redundant epigenetic silencing pathways.

Cytosine methylation is critical in mammalian development and plays a role in diverse biologic processes such as genomic imprinting, X chromosome inactivation, and silencing of repeat elements. Several factors regulate DNA methylation in early embryogenesis, but their precise role in the establishment of DNA methylation at a given site remains unclear. We have generated a comprehensive methylation map in fibroblasts derived from the murine DNA methylation mutant Hells(-/-) (helicase, lymphoid specific, also known as LSH). It has been previously shown that HELLS can influence de novo methylation of retroviral sequences and endogenous genes. Here, we describe that HELLS controls cytosine methylation in a nuclear compartment that is in part defined by lamin B1 attachment regions. Despite widespread loss of cytosine methylation at regulatory sequences, including promoter regions of protein-coding genes and noncoding RNA genes, overall relative transcript abundance levels in the absence of HELLS are similar to those in wild-type cells. A subset of promoter regions shows increases of the histone modification H3K27me3, suggesting redundancy of epigenetic silencing mechanisms. Furthermore, HELLS modulates CG methylation at all classes of repeat elements and is critical for repression of a subset of repeat elements. Overall, we provide a detailed analysis of gene expression changes in relation to DNA methylation alterations, which contributes to our understanding of the biological role of cytosine methylation.

The ATP binding site of the chromatin remodeling homolog Lsh is required for nucleosome density and de novo DNA methylation at repeat sequences.

Lsh, a chromatin remodeling protein of the SNF2 family, is critical for normal heterochromatin structure. In particular, DNA methylation at repeat elements, a hallmark of heterochromatin, is greatly reduced in Lsh(-/-) (KO) cells. Here, we examined the presumed nucleosome remodeling activity of Lsh on chromatin in the context of DNA methylation. We found that dynamic CG methylation was dependent on Lsh in embryonic stem cells. Moreover, we demonstrate that ATP function is critical for de novo methylation at repeat sequences. The ATP binding site of Lsh is in part required to promote stable association of the DNA methyltransferase 3b with the repeat locus. By performing nucleosome occupancy assays, we found distinct nucleosome occupancy in KO ES cells compared to WT ES cells after differentiation. Nucleosome density was restored to wild-type level by re-expressing wild-type Lsh but not the ATP mutant in KO ES cells. Our results suggest that ATP-dependent nucleosome remodeling is the primary molecular function of Lsh, which may promote de novo methylation in differentiating ES cells.

CG hypomethylation in Lsh-/- mouse embryonic fibroblasts is associated with de novo H3K4me1 formation and altered cellular plasticity.

DNA methylation patterns are established in early embryogenesis and are critical for cellular differentiation. To investigate the role of CG methylation in potential enhancer formation, we assessed H3K4me1 modification in murine embryonic fibroblasts (MEFs) derived from the DNA methylation mutant Lsh(-/-) mice. We report here de novo formation of putative enhancer elements at CG hypomethylated sites that can be dynamically altered. We found a subset of differentially enriched H3K4me1 regions clustered at neuronal lineage genes and overlapping with known cis-regulatory elements present in brain tissue. Reprogramming of Lsh(-/-) MEFs into induced pluripotent stem (iPS) cells leads to increased neuronal lineage gene expression of premarked genes and enhanced differentiation potential of Lsh(-/-) iPS cells toward the neuronal lineage pathway compared with WT iPS cells in vitro and in vivo. The state of CG hypomethylation and H3K4me1 enrichment is partially maintained in Lsh(-/-) iPS cells. The acquisition of H3K27ac and activity of subcloned fragments in an enhancer reporter assay indicate functional activity of several of de novo H3K4me1-marked sequences. Our results suggest a functional link of H3K4me1 enrichment at CG hypomethylated sites, enhancer formation, and cellular plasticity.

Diesel exhaust particulate matter induces GC-1 spg cells oxidative stress by KEAP1-NRF2 pathway and inhibition of ATP5α1 S-sulfhydration.

Diesel exhaust particle (DEP) exposure induces a variety of toxicological effects through oxidative stress and inflammation responses. This research investigated the mechanisms underlying DEP-induced GC-1spg cells oxidative stress by examining ROS accumulation, antioxidant defense systems activation, mitochondrial dysfunction, and the Nrf2/Keap1/HO-1 pathway response. Subsequently, we further evaluated the ATP levels, ATP5α synthase activity and ATP5α synthase S-sulfhydrated modification in DEP-exposed GC-1 spg cells. The results showed that DEP exposure significantly inhibited cell proliferation and viability, increased intracellular ROS production, decreased MMP, down-regulated antioxidant capacity, activated the Nrf2/Keap1/HO-1 pathway. However, DEP-induced oxidative stress was partially alleviated by GSH and exogenous H2S. In addition, DEP exposure induced ATP depletion and ATP5α synthase inactivity in GC-1 spg cells, accompanied by ATP5α synthase S-sulfhydrated modification. In conclusion, our research showed that DEP may incapacitate mitochondria through oxidative stress injury, leading to GC-1 spg cells oxidative stress. This process may be associated with the reduction of ATP5α1 S-sulfhydrated modification. It provides a new perspective for the research of the mechanism related to male reproductive toxicity due to air pollution.

UHRF1/DNMT1-MZF1 axis-modulated intragenic site-specific CpGI methylation confers divergent expression and opposing functions of PRSS3 isoforms in lung cancer.

As confusion mounts over RNA isoforms involved in phenotypic plasticity, aberrant CpG methylation-mediated disruption of alternative splicing is increasingly recognized as a driver of intratumor heterogeneity (ITH). Protease serine 3 (PRSS3), possessing four splice variants (PRSS3-SVs; PRSS3-V1-V4), is an indispensable trypsin that shows paradoxical effects on cancer development. Here, we found that PRSS3 transcripts and their isoforms were divergently expressed in lung cancer, exhibiting opposing functions and clinical outcomes, namely, oncogenic PRSS3-V1 and PRSS3-V2 versus tumor-suppressive PRSS3-V3, by targeting different downstream genes. We identified an intragenic CpG island (iCpGI) in PRSS3. Hypermethylation of iCpGI was mediated by UHRF1/DNMT1 complex interference with the binding of myeloid zinc finger 1 (MZF1) to regulate PRSS3 transcription. The garlic-derived compound diallyl trisulfide cooperated with 5-aza-2'-deoxycytidine to exert antitumor effects in lung adenocarcinoma cells through site-specific iCpGI demethylation specifically allowing MZF1 to upregulate PRSS3-V3 expression. Epigenetic silencing of PRSS3-V3 via iCpGI methylation (iCpGIm) in BALF and tumor tissues was associated with early clinical progression in patients with lung cancer but not in those with squamous cell carcinoma or inflammatory disease. Thus, UHRF1/DNMT1-MZF1 axis-modulated site-specific iCpGIm regulates divergent expression of PRSS3-SVs, conferring nongenetic functional ITH, with implications for early detection of lung cancer and targeted therapies.

Germinal center output is sustained by HELLS-dependent DNA-methylation-maintenance in B cells.

HELLS/LSH (Helicase, Lymphoid Specific) is a SNF2-like chromatin remodelling protein involved in DNA methylation. Its loss-of-function in humans causes humoral immunodeficiency, called ICF4 syndrome (Immunodeficiency, Centromeric Instability, Facial anomalies). Here we show by our newly generated B-cell-specific Hells conditional knockout mouse model that HELLS plays a pivotal role in T-dependent B-cell responses. HELLS deficiency induces accelerated decay of germinal center (GC) B cells and impairs the generation of high affinity memory B cells and circulating antibodies. Mutant GC B cells undergo dramatic DNA hypomethylation and massive de-repression of evolutionary recent retrotransposons, which surprisingly does not directly affect their survival. Instead, they prematurely upregulate either memory B cell markers or the transcription factor ATF4, which is driving an mTORC1-dependent metabolic program typical of plasma cells. Treatment of wild type mice with a DNMT1-specific inhibitor phenocopies the accelerated kinetics, thus pointing towards DNA-methylation maintenance by HELLS being a crucial mechanism to fine-tune the GC transcriptional program and enable long-lasting humoral immunity.

An Inflammatory Checkpoint Generated by IL1RN Splicing Offers Therapeutic Opportunity for KRAS-Mutant Intrahepatic Cholangiocarcinoma.

KRAS mutations are causally linked to protumor inflammation and are identified as driving factors in tumorigenesis. Here, using multiomics data gathered from a large set of patients, we showed that KRAS mutation was associated with a specific landscape of alternative mRNA splicing that connected to myeloid inflammation in intrahepatic cholangiocarcinoma (iCCA). Then, we identified a negative feedback mechanism in which the upregulation of interleukin 1 receptor antagonist (IL1RN)-201/203 due to alternative splicing confers vital anti-inflammatory effects in KRAS-mutant iCCA. In KRAS-mutant iCCA mice, both IL1RN-201/203 upregulation and anakinra treatment ignited a significant antitumor immune response by altering neutrophil recruitment and phenotypes. Furthermore, anakinra treatment synergistically enhanced anti-PD-1 therapy to activate intratumoral GZMB+ CD8+ T cells in KRAS-mutant iCCA mice. Clinically, we found that high IL1RN-201/203 levels in patients with KRAS-mutant iCCA were significantly associated with superior response to anti-PD-1 immunotherapy. SIGNIFICANCE: This work describes a novel inflammatory checkpoint mediated by IL1RN alternative splicing variants that may serve as a promising basis to develop therapeutic options for KRAS-mutant iCCA and other cancers. This article is featured in Selected Articles from This Issue, p. 2109.

NRSF/REST is required for gastrulation and neurogenesis during zebrafish development.

Repressor element 1-silencing transcription factor (REST) was recognized as a transcription suppressor regulating nervous system differentiation. However, the role of REST during early development has not been clarified. We cloned the zebrafish homolog of human REST. Real-time polymerase chain reaction results showed that zebrafish REST mRNA was both maternal and zygotic with the higher expression level from blastula to the late segmentation period. Whole-mount in situ hybridization showed that REST was strongly expressed in the blastoderm since dome stage and dynamically expressed mainly in developing brain, especially in the border of the brain subdivisions in early segmentation period. Knockdown of REST using translation blocking morpholino (MO-tra) technique resulted in gastrulation delay or even blockage, and subsequently led to embryo lethality by early segmentation period with deficient neurogenesis. However, splicing blocking morpholino for REST did not show obviously abnormal phenotype until 48 hpf (hours post-fertilization), indicating that maternal REST was an important regulator for gastrulation. Further study revealed that the abnormal development in MO-tra morphants was at least partly due to the dysfunction of protein transportation from the yolk to the blastoderm. Our results showed that REST (especially maternal supplied REST) was required for gastrulation and neurogenesis during zebrafish early embryogenesis.

The Monocyte-to-Lymphocyte Ratio Predicts Acute Kidney Injury After Acute Hemorrhagic Stroke.

Objectives: Acute kidney injury (AKI) is a serious complication of acute hemorrhagic stroke (AHS). Early detection and early treatment are crucial for patients with AKI. We conducted a study to analyze the role of the monocyte-to-lymphocyte ratio (MLR) in predicting the development of AKI after AHS. Methods: This retrospective observational study enrolled all subjects with AHS who attended the neurosurgical intensive care unit (NSICU) at the First Affiliated University of South China between 2018 and 2021. Patient demographics, laboratory data, treatment details, and clinical outcomes were recorded. Results: Of the 771 enrolled patients, 180 (23.3%) patients developed AKI. Compared to patients without AKI, those with AKI had a higher MLR and the neutrophil-lymphocyte ratio (NLR) at admission (P < 0.001). The MLR and the NLR at admission were associated with an increased AKI risk, with odds ratios (ORs) of 8.27 (95% CI: 4.23, 16.17, p < 0.001) and 1.17 (95% CI: 1.12, 1.22, p < 0.001), respectively. The receiver operating characteristic curve (ROC) analysis was conducted to analyze the ability of the MLR and NLR to predict AKI, and the areas under the curve (AUCs) of the MLR and the NLR were 0.73 (95% CI: 0.69, 0.77, p < 0.001) and 0.67 (95% CI: 0.62, 0.72, p < 0.001), with optimal cutoff values of 0.5556 and 11.65, respectively. The MLR and the NLR at admission were associated with an increased in-hospital mortality risk, with ORs of 3.13 (95% CI: 1.08, 9.04) and 1.07 (95% CI: 1.00, 1.14), respectively. The AUCs of the MLR and the NLR for predicting in-hospital mortality were 0.62 (95% CI: 0.54, 0.71, p = 0.004) and 0.52 (95% CI: 0.43, 0.62, p = 0.568), respectively. The optimal cutoff value for the MLR was 0.7059, with a sensitivity of 51% and a specificity of 73.3%. Conclusions: MLR and NLR measurements in patients with AHS at admission could be valuable tools for identifying patients at high risk of early AKI. The MLR was positively associated with in-hospital mortality and the NLR showed a weak ability for the prediction of in-hospital mortality.

CpG Site-Specific Methylation-Modulated Divergent Expression of PRSS3 Transcript Variants Facilitates Nongenetic Intratumor Heterogeneity in Human Hepatocellular Carcinoma.

Background: Hepatocellular carcinoma (HCC) is one of the most lethal human tumors with extensive intratumor heterogeneity (ITH). Serine protease 3 (PRSS3) is an indispensable member of the trypsin family and has been implicated in the pathogenesis of several malignancies, including HCC. However, the paradoxical effects of PRSS3 on carcinogenesis due to an unclear molecular basis impede the utilization of its biomarker potential. We hereby explored the contribution of PRSS3 transcripts to tumor functional heterogeneity by systematically dissecting the expression of four known splice variants of PRSS3 (PRSS3-SVs, V1~V4) and their functional relevance to HCC. Methods: The expression and DNA methylation of PRSS3 transcripts and their associated clinical relevance in HCC were analyzed using several publicly available datasets and validated using qPCR-based assays. Functional experiments were performed in gain- and loss-of-function cell models, in which PRSS3 transcript constructs were separately transfected after deleting PRSS3 expression by CRISPR/Cas9 editing. Results: PRSS3 was aberrantly differentially expressed toward bipolarity from very low (PRSS3Low ) to very high (PRSS3High ) expression across HCC cell lines and tissues. This was attributable to the disruption of PRSS3-SVs, in which PRSS3-V2 and/or PRSS3-V1 were dominant transcripts leading to PRSS3 expression, whereas PRSS3-V3 and -V4 were rarely or minimally expressed. The expression of PRSS3-V2 or -V1 was inversely associated with site-specific CpG methylation at the PRSS3 promoter region that distinguished HCC cells and tissues phenotypically between hypermethylated low-expression (mPRSS3-SVLow ) and hypomethylated high-expression (umPRSS3-SVHigh ) groups. PRSS3-SVs displayed distinct functions from oncogenic PRSS3-V2 to tumor-suppressive PRSS3-V1, -V3 or PRSS3-V4 in HCC cells. Clinically, aberrant expression of PRSS3-SVs was translated into divergent relevance in patients with HCC, in which significant epigenetic downregulation of PRSS3-V2 was seen in early HCC and was associated with favorable patient outcome. Conclusions: These results provide the first evidence for the transcriptional and functional characterization of PRSS3 transcripts in HCC. Aberrant expression of divergent PRSS3-SVs disrupted by site-specific CpG methylation may integrate the effects of oncogenic PRSS3-V2 and tumor-suppressive PRSS3-V1, resulting in the molecular diversity and functional plasticity of PRSS3 in HCC. Dysregulated expression of PRSS3-V2 by site-specific CpG methylation may have potential diagnostic value for patients with early HCC.

Lyzl4, a novel mouse sperm-related protein, is involved in fertilization.

The role of Chicken-type (c-type) lysozyme, a prototype lysozyme, in immunity has been characterized in many organisms. In this study, we cloned a novel c-type lysozyme-like gene, Lyzl4, which was located on mouse chromosome 9F4 and encoded 145 amino acids with a putative signal peptide and a protease cleavage site. The mature recombinant Lyzl4 protein expressed in yeast did not show the bacteriolytic activity. Sequence alignment analysis demonstrated that 3 of the 20 invariant residues in c-type lysozymes were changed in Lyzl4. One of the 'changed' amino acids (D52G) is located in the catalytic domain. Lyzl4 mRNA was selectively expressed in testis and epididymis in adult mice, with varying expression level across different developmental stages. High level of Lyzl4 protein was found on the spermatozoa of acrosomal region and principal piece of tail. Immuno-neutralization of Lyzl4 protein in spermatozoa with its specific antibody significantly decreased in vitro fertilization percentage in a dose-dependent manner, suggesting that Lyzl4 might be important for fertilization.

A Promising Intracellular Protein-Degradation Strategy: TRIMbody-Away Technique Based on Nanobody Fragment.

Most recently, a technology termed TRIM-Away has allowed acute and rapid destruction of endogenous target proteins in cultured cells using specific antibodies and endogenous/exogenous tripartite motif 21 (TRIM21). However, the relatively large size of the full-size mAbs (150 kDa) results in correspondingly low tissue penetration and inaccessibility of some sterically hindered epitopes, which limits the target protein degradation. In addition, exogenous introduction of TRIM21 may cause side effects for treated cells. To tackle these limitations, we sought to replace full-size mAbs with the smaller format of antibodies, a nanobody (VHH, 15 kDa), and construct a new type of fusion protein named TRIMbody by fusing the nanobody and RBCC motif of TRIM21. Next, we introduced enhanced green fluorescent protein (EGFP) as a model substrate and generated αEGFP TRIMbody using a bispecific anti-EGFP (αEGFP) nanobody. Remarkably, inducible expression of αEGFP TRIMbody could specifically degrade intracellular EGFP in HEK293T cells in a time-dependent manner. By treating cells with inhibitors, we found that intracellular EGFP degradation by αEGFP TRIMbody relies on both ubiquitin-proteasome and autophagy-lysosome pathways. Taken together, these results suggested that TRIMbody-Away technology could be utilized to specifically degrade intracellular protein and could expand the potential applications of degrader technologies.

The epigenetic regulator LSH maintains fork protection and genomic stability via MacroH2A deposition and RAD51 filament formation.

The Immunodeficiency Centromeric Instability Facial Anomalies (ICF) 4 syndrome is caused by mutations in LSH/HELLS, a chromatin remodeler promoting incorporation of histone variant macroH2A. Here, we demonstrate that LSH depletion results in degradation of nascent DNA at stalled replication forks and the generation of genomic instability. The protection of stalled forks is mediated by macroH2A, whose knockdown mimics LSH depletion and whose overexpression rescues nascent DNA degradation. LSH or macroH2A deficiency leads to an impairment of RAD51 loading, a factor that prevents MRE11 and EXO1 mediated nascent DNA degradation. The defect in RAD51 loading is linked to a disbalance of BRCA1 and 53BP1 accumulation at stalled forks. This is associated with perturbed histone modifications, including abnormal H4K20 methylation that is critical for BRCA1 enrichment and 53BP1 exclusion. Altogether, our results illuminate the mechanism underlying a human syndrome and reveal a critical role of LSH mediated chromatin remodeling in genomic stability.

LSH mediates gene repression through macroH2A deposition.

The human Immunodeficiency Centromeric Instability Facial Anomalies (ICF) 4 syndrome is a severe disease with increased mortality caused by mutation in the LSH gene. Although LSH belongs to a family of chromatin remodeling proteins, it remains unknown how LSH mediates its function on chromatin in vivo. Here, we use chemical-induced proximity to rapidly recruit LSH to an engineered locus and find that LSH specifically induces macroH2A1.2 and macroH2A2 deposition in an ATP-dependent manner. Tethering of LSH induces transcriptional repression and silencing is dependent on macroH2A deposition. Loss of LSH decreases macroH2A enrichment at repeat sequences and results in transcriptional reactivation. Likewise, reduction of macroH2A by siRNA interference mimicks transcriptional reactivation. ChIP-seq analysis confirmed that LSH is a major regulator of genome-wide macroH2A distribution. Tethering of ICF4 mutations fails to induce macroH2A deposition and ICF4 patient cells display reduced macroH2A deposition and transcriptional reactivation supporting a pathogenic role for altered marcoH2A deposition. We propose that LSH is a major chromatin modulator of the histone variant macroH2A and that its ability to insert marcoH2A into chromatin and transcriptionally silence is disturbed in the ICF4 syndrome.

The chromatin remodeling protein Lsh alters nucleosome occupancy at putative enhancers and modulates binding of lineage specific transcription factors.

Dynamic regulation of chromatin accessibility is a key feature of cellular differentiation during embryogenesis, but the precise factors that control access to chromatin remain largely unknown. Lsh/HELLS is critical for normal development and mutations of Lsh in human cause the ICF (Immune deficiency, Centromeric instability, Facial anomalies) syndrome, a severe immune disorder with multiple organ deficiencies. We report here that Lsh, previously known to regulate DNA methylation level, has a genome wide chromatin remodeling function. Using micrococcal nuclease (MNase)-seq analysis, we demonstrate that Lsh protects MNase accessibility at transcriptional regulatory regions characterized by DNase I hypersensitivity and certain histone 3 (H3) tail modifications associated with enhancers. Using an auxin-inducible degron system, allowing proteolytical degradation of Lsh, we show that Lsh mediated changes in nucleosome occupancy are independent of DNA methylation level and are characterized by reduced H3 occupancy. While Lsh mediated nucleosome occupancy prevents binding sites for transcription factors in wild type cells, depletion of Lsh leads to an increase in binding of ectopically expressed tissue specific transcription factors to their respective binding sites. Our data suggests that Lsh mediated chromatin remodeling can modulate nucleosome positioning at a subset of putative enhancers contributing to the preservation of cellular identity through regulation of accessibility.

Functional imaging of interleukin 1 beta expression in inflammatory process using bioluminescence imaging in transgenic mice.

BACKGROUND: Interleukin 1 beta (IL-1beta) plays an important role in a number of chronic and acute inflammatory diseases. To understand the role of IL-1beta in disease processes and develop an in vivo screening system for anti-inflammatory drugs, a transgenic mouse line was generated which incorporated the transgene firefly luciferase gene driven by a 4.5-kb fragment of the human IL-1beta gene promoter. Luciferase gene expression was monitored in live mice under anesthesia using bioluminescence imaging in a number of inflammatory disease models. RESULTS: In a LPS-induced sepsis model, dramatic increase in luciferase activity was observed in the mice. This transgene induction was time dependent and correlated with an increase of endogenous IL-1beta mRNA and pro-IL-1beta protein levels in the mice. In a zymosan-induced arthritis model and an oxazolone-induced skin hypersensitivity reaction model, luciferase expression was locally induced in the zymosan injected knee joint and in the ear with oxazolone application, respectively. Dexamethasone suppressed the expression of luciferase gene both in the acute sepsis model and in the acute arthritis model. CONCLUSION: Our data suggest that the transgenic mice model could be used to study transcriptional regulation of the IL-1beta gene expression in the inflammatory process and evaluation the effect of anti-inflammatory drug in vivo.

Improved method to raise polyclonal antibody using enhanced green fluorescent protein transgenic mice.

Recombinant fusion protein is widely used as an antigen to raise antibodies against the epitope of a target protein. However, the concomitant anticarrier antibody in resulting antiserum reduces the production of the desired antibody and brings about unwanted non-specific immune reactions. It is proposed that the carrier protein transgenic animal could be used to solve this problem. To validate this hypothesis, enhanced green fluorescent protein (EGFP) transgenic mice were produced. By immunizing the mice with fusion protein His6HAtag-EGFP, we showed that the antiserum from the transgenic mice had higher titer antibody against His6HA tag and lower titer antibody against EGFP compared with that from wild-type mice. Therefore, this report describes an improved method to raise high titer antipeptide polyclonal antibody using EGFP transgenic mice that could have application potential in antibody preparation.

Tethering of Lsh at the Oct4 locus promotes gene repression associated with epigenetic changes.

Lsh is a chromatin remodeling factor that regulates DNA methylation and chromatin function in mammals. The dynamics of these chromatin changes and whether they are directly controlled by Lsh remain unclear. To understand the molecular mechanisms of Lsh chromatin controlled regulation of gene expression, we established a tethering system that recruits a Gal4-Lsh fusion protein to an engineered Oct4 locus through Gal4 binding sites in murine embryonic stem (ES) cells. We examined the molecular epigenetic events induced by Lsh binding including: histone modification, DNA methylation and chromatin accessibility to determine nucleosome occupancy before and after embryonic stem cell differentiation. Our results indicate that Lsh assists gene repression upon binding to the Oct4 promoter region. Furthermore, we detected less chromatin accessibility and reduced active histone modifications at the tethered site in undifferentiated ES, while GFP reporter gene expression and DNA methylation patterns remained unchanged at this stage. Upon differentiation, association of Lsh promotes transcriptional repression of the reporter gene accompanied by the increase of repressive histone marks and a gain of DNA methylation at distal and proximal Oct4 enhancer sites. Taken together, this approach allowed us to examine Lsh mediated epigenetic regulation as a dynamic process and revealed chromatin accessibility changes as the primary consequence of Lsh function.

Lsh/HELLS regulates self-renewal/proliferation of neural stem/progenitor cells.

Epigenetic mechanisms are known to exert control over gene expression and determine cell fate. Genetic mutations in epigenetic regulators are responsible for several neurologic disorders. Mutations of the chromatin remodeling protein Lsh/HELLS can cause the human Immunodeficiency, Centromere instability and Facial anomalies (ICF) syndrome, which is associated with neurologic deficiencies. We report here a critical role for Lsh in murine neural development. Lsh depleted neural stem/progenitor cells (NSPCs) display reduced growth, increases in apoptosis and impaired ability of self-renewal. RNA-seq analysis demonstrates differential gene expression in Lsh-/- NSPCs and suggests multiple aberrant pathways. Concentrating on specific genomic targets, we show that ablation of Lsh alters epigenetic states at specific enhancer regions of the key cell cycle regulator Cdkn1a and the stem cell regulator Bmp4 in NSPCs and alters their expression. These results suggest that Lsh exerts epigenetic regulation at key regulators of neural stem cell fate ensuring adequate NSPCs self-renewal and maintenance during development.