Discovery of benzo[d]isothiazole derivatives as novel scaffold inhibitors targeting the programmed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1) interaction through "ring fusion" strategy.

The inhibition of programmed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1) interaction by monoclonal antibodies (mAbs) has achieved promising outcomes in cancer immunotherapy. Due to the inherent deficiencies of mAbs drugs, such as high cost of treatment, immunogenicity, poor pharmacokinetics and penetration of solid tumors, researchers are encouraged to develop small molecule inhibitors, to overcome mAbs drugs' deficiencies and change the situation where small molecule drugs are not available on the market. Herein, we reported a series of benzo[d]isothiazole derivatives targeting the PD-1/PD-L1 interaction through "ring fusion" strategy using BMS-202 as a starting point. Among them, compound D7 exhibited the best inhibitory activity with an IC50 value of 5.7 nM by homogeneous time-resolved fluorescence (HTRF) binding assay. In immunotoxicity analysis, D7 showed low cytotoxicity to Jurkat T cells in CCK-8 assay compared to BMS-202. The binding mode between D7 and PD-L1 protein was explored by molecular docking and molecular dynamics (MD) simulations, which revealed crucial chemical groups, such as biphenyl group interacting with Ile54A, Tyr56A, Met115A, Ala121A, Ile54B, Met115B, Ala121B and Tyr123B by hydrophobic interactions, bromobenzene moiety forming π-π stacking interaction with Tyr56B, as well as l-serine moiety forming hydrogen bond (H-bond) and salt bridge interactions with Asp122A and Lys124A. Furthermore, molecular modeling studies showed that D7 is likely to bind to the FA8 (fatty acid 8) binding site of human serum albumin (HSA). Taken together, D7 significantly inhibits the PD-1/PD-L1 interaction with low cytotoxicity, indicating that D7 is a promising starting point for further drug development in cancer immunotherapy.

Study of Environmental Enteropathy and Malnutrition (SEEM) in Pakistan: protocols for biopsy based biomarker discovery and validation.

BACKGROUND: Environmental Enteropathy (EE), characterized by alterations in intestinal structure, function, and immune activation, is believed to be an important contributor to childhood undernutrition and its associated morbidities, including stunting. Half of all global deaths in children < 5 years are attributable to under-nutrition, making the study of EE an area of critical priority. METHODS: Community based intervention study, divided into two sub-studies, 1) Longitudinal analyses and 2) Biopsy studies for identification of EE features via omics analyses. Birth cohorts in Matiari, Pakistan established: moderately or severely malnourished (weight for height Z score (WHZ) < - 2) children, and well-nourished (WHZ > 0) children. Blood, urine, and fecal samples, for evaluation of potential biomarkers, will be collected at various time points from all participants (longitudinal analyses). Participants will receive appropriate educational and nutritional interventions; non-responders will undergo further evaluation to determine eligibility for further workup, including upper gastrointestinal endoscopy. Histopathological changes in duodenal biopsies will be compared with duodenal biopsies obtained from USA controls who have celiac disease, Crohn's disease, or who were found to have normal histopathology. RNA-Seq will be employed to characterize mucosal gene expression across groups. Duodenal biopsies, luminal aspirates from the duodenum, and fecal samples will be analyzed to define microbial community composition (omic analyses). The relationship between histopathology, mucosal gene expression, and community configuration will be assessed using a variety of bioinformatic tools to gain better understanding of disease pathogenesis and to identify mechanism-based biomarkers. Ethical review committees at all collaborating institutions have approved this study. All results will be made available to the scientific community. DISCUSSION: Operational and ethical constraints for safely obtaining intestinal biopsies from children in resource-poor settings have led to a paucity of human tissue-based investigations to understand and reverse EE in vulnerable populations. Furthermore, EE biomarkers have rarely been correlated with gold standard histopathological confirmation. The Study of Environmental Enteropathy and Malnutrition (SEEM) is designed to better understand the pathophysiology, predictors, biomarkers, and potential management strategies of EE to inform strategies to eradicate this debilitating pathology and accelerate progress towards the 2030 Sustainable Development Goals. TRIAL REGISTRATION: Retrospectively registered; clinicaltrials.gov ID NCT03588013 .

Cell-autonomous activation of Hedgehog signaling inhibits brown adipose tissue development.

Although recent studies have shown that brown adipose tissue (BAT) arises from progenitor cells that also give rise to skeletal muscle, the developmental signals that control the formation of BAT remain largely unknown. Here, we show that brown preadipocytes possess primary cilia and can respond to Hedgehog (Hh) signaling. Furthermore, cell-autonomous activation of Hh signaling blocks early brown-preadipocyte differentiation, inhibits BAT formation in vivo, and results in replacement of neck BAT with poorly differentiated skeletal muscle. Finally, we show that Hh signaling inhibits BAT formation partially through up-regulation of chicken ovalbumin upstream promoter transcription factor II (COUP-TFII). Taken together, our studies uncover a previously unidentified role for Hh as an inhibitor of BAT development.

Major epigenetic development distinguishing neuronal and non-neuronal cells occurs postnatally in the murine hypothalamus.

Prenatal and early postnatal environment can persistently alter one's risk of obesity. Environmental effects on hypothalamic developmental epigenetics constitute a likely mechanism underlying such 'developmental programming' of energy balance regulation. To advance our understanding of these processes, it is essential to develop approaches to disentangle the cellular and regional heterogeneity of hypothalamic developmental epigenetics. We therefore performed genome-scale DNA methylation profiling in hypothalamic neurons and non-neuronal cells at postnatal day 0 (P0) and P21 and found, surprisingly, that most of the DNA methylation differences distinguishing these two cell types are established postnatally. In particular, neuron-specific increases in DNA methylation occurred extensively at genes involved in neuronal development. Quantitative bisulfite pyrosequencing verified our methylation profiling results in all 15 regions examined, and expression differences were associated with DNA methylation at several genes. We also identified extensive methylation differences between the arcuate (ARH) and paraventricular nucleus of the hypothalamus (PVH). Integrating these two data sets showed that genomic regions with PVH versus ARH differential methylation strongly overlap with those undergoing neuron-specific increases from P0 to P21, suggesting that these developmental changes occur preferentially in either the ARH or PVH. In particular, neuron-specific methylation increases at the 3' end of Shh localized to the ARH and were positively associated with gene expression. Our data indicate a key role for DNA methylation in establishing the gene expression potential of diverse hypothalamic cell types, and provide the novel insight that early postnatal life is a critical period for cell type-specific epigenetic development in the murine hypothalamus.

Comparison and quantitative verification of mapping algorithms for whole-genome bisulfite sequencing.

Coupling bisulfite conversion with next-generation sequencing (Bisulfite-seq) enables genome-wide measurement of DNA methylation, but poses unique challenges for mapping. However, despite a proliferation of Bisulfite-seq mapping tools, no systematic comparison of their genomic coverage and quantitative accuracy has been reported. We sequenced bisulfite-converted DNA from two tissues from each of two healthy human adults and systematically compared five widely used Bisulfite-seq mapping algorithms: Bismark, BSMAP, Pash, BatMeth and BS Seeker. We evaluated their computational speed and genomic coverage and verified their percentage methylation estimates. With the exception of BatMeth, all mappers covered >70% of CpG sites genome-wide and yielded highly concordant estimates of percentage methylation (r(2) ≥ 0.95). Fourfold variation in mapping time was found between BSMAP (fastest) and Pash (slowest). In each library, 8-12% of genomic regions covered by Bismark and Pash were not covered by BSMAP. An experiment using simulated reads confirmed that Pash has an exceptional ability to uniquely map reads in genomic regions of structural variation. Independent verification by bisulfite pyrosequencing generally confirmed the percentage methylation estimates by the mappers. Of these algorithms, Bismark provides an attractive combination of processing speed, genomic coverage and quantitative accuracy, whereas Pash offers considerably higher genomic coverage.

[An Incoherent Broadband Optical Cavity Spectroscopy for Measuring Weak Absorption Cross Section of Sulfur Dioxide].

As a highly sensitive detection technology, incoherent broadband cavity enhanced absorption spectroscopy (IBBCEAS) have successfully measured a variety of trace gases. According to the principle of cavity enhanced absorption spectroscopy, if the accurate concentration of the target gas, the curve of the mirror reflectance, effective absorption path length, the light intensity of the absorbing gas and non-absorbing gas are known, the absorption cross section of the absorption gas can be measured. The accurate measurements of absorption cross section are necessary for satellite retrievals of atmospheric trace gases and the atmospheric research. This paper describes an incoherent broadband cavity enhanced absorption spectroscopy(IBBCEAS) instrument with 365 nm LED as the light source for measuring absorption cross section of SO2 from 357 to 385 nm which is arising from the spin-forbidden a ³B1--X¹A1 transition. In comparison to the literature absorption cross section of SO2, and correlation coefficient r is 0.997 3. The result shows the potential of the IBBCEAS system for measuring weak absorption cross section.

[The Detection of Atmospheric HONO and NO2 with Fiber Coupling Long-Path Differential Optical Absorption Spectroscopy System].

A new type of fiber coupling Long-Path Differential Optical Absorption Spectroscopy System(LP-DOAS) based on schmidt - cassegrain telescope was introduced in detail in this paper and it was applied to the accurate measurement of the actual atmospheric HONO and NO2. This measuring system simplified the structure of traditional LP-DOAS system, combining with the design of optical fiber coupling.It made full use of the telescope primary mirror's effective area. The effects of the offset, dark current and telescope stray light to the new LP-DOAS system were discussed in this paper; On a clear day, the ratio between telescope stray light and the optical intensities was less than 1%. To verify the accurate of the new LP-DOAS system, the atmospheric NO2 were simultaneously measured with the new LP-DOAS system and traditional LP-DOAS system. The correlation coefficient R2 was up to 0.968. The observation of atmospheric HONO was carried out by using the fiber coupling LP-DOAS in Gucheng, Hebei Province, China, and the detection limit (2σ) of HONO and NO2 was 84.2 and 144.6 ppt , respectively, with 2 490 m path length and the average time resolution of about 30 s. In the whole measurement in Gucheng, the maximum of HONO and NO2 were 3.2 and 37.8 ppb, respectively, and the minimum were both under the detection limits; the ratio of HONO/NO2 at night was calculated.

[Cruise Observation of SO2, NO2 and Benzene with Mobile Portable DOAS in the Industrial Park].

The self-developed portable DOAS instrument based on differential optical absorption spectroscopy(DOAS) and composed of optical fiber spectrograph and multiple-pass cell was introduced. The standard gases of SO2 and NO2 were employed to test the accuracy and stability of the system, and then cruise observation of SO2, NO2 and benzene was carried out using the system in Tongling industrial park. During the entire period, the polluted gases showed high concentrations near the contaminated areas and the maximum concentrations of SO2, NO2 and benzene were 5 023.2, 2 195.2 and 162.5 µg·m-3, respectively. With 12.6 m optical path, the detection limits of SO2, NO2 and benzene were 67.0, 169.9, 30.6 µg·m-3, respectively. The portable DOAS system provides a convenient and effective technique for industrial park about emergency and supervisory monitoring and evaluation of gas leakage and fugitive emissions of gaseous pollutants.

Regulation of AURKC expression by CpG island methylation in human cancer cells.

AURKC, a member of the Aurora kinase gene family, is highly expressed in testis but is either moderately expressed or repressed in most somatic cells. Varying expression of AURKC has been observed in human cancers, but the underlying mechanisms of differential expression have been investigated only to a limited extent. We investigated the role of promoter CpG methylation in the regulation of AURKC gene expression in human cancer cells, in relation to a recently reported AURKC transcription repressor PLZF/ZBTB16, implicated in transformation and tumorigenesis. AURKC and PLZF/ZBTB16 expression profiles were investigated in reference to CpG methylation status on the AURKC promoter experimentally, and also in The Cancer Genome Atlas (TCGA) dataset involving multiple cancer types. AURKC promoter showed dense to moderate hypermethylation correlating with low to moderate expression of the gene in normal somatic cells and cancer cell lines, while testis with high expression revealed marked hypo-methylation. Treatment with the demethylating agent, 5-aza-dC, but not the histone deacetylase (HDAC) inhibitor, TSA, led to elevated expression in cancer cell lines, indicating that promoter DNA methylation negatively regulates AURKC expression. High expression of PLZF in PLZF-transfected cells treated with 5-aza-dC only partially repressed expression of AURKC despite 5-aza-dC also inducing elevated PLZF expression. Analyses of the TCGA data showed differential expression of AURKC in multiple cancer types and stronger correlation of AURKC expression with CpG methylation compared to PLZF levels. These findings demonstrate that differential promoter CpG methylation is an important mechanism regulating AURKC expression in cancer cells.

Repetitive elements and enforced transcriptional repression co-operate to enhance DNA methylation spreading into a promoter CpG-island.

Repression of many tumor suppressor genes in cancer is concurrent with aberrantly increased DNA methylation levels at promoter CpG islands (CGIs). About one-fourth of empirically defined human promoters are surrounded by or contain clustered repetitive elements. It was previously observed that a sharp transition of methylation exists between highly methylated repetitive elements and unmethylated promoter-CGIs in normal tissues. The factors that lead to aberrant CGI hypermethylation in cancer remain poorly understood. Here, we established a site-specific integration system with enforced local transcriptional repression in colorectal cancer cells and monitored the occurrence of initial de novo methylation at specific CG sites adjacent to the CGI of the INSL6 promoter, which could be accelerated by binding a KRAB-containing transcriptional factor. Additional repetitive elements from P16 and RIL (PDLIM4), if situated adjacent to the promoter of INSL6, could confer DNA methylation spreading into the CGI particularly in the setting of KRAB-factor binding. However, a repressive chromatin alone was not sufficient to initiate DNA methylation, which required specific DNA sequences and was integration-site (and/or cell-line) specific. Overall, these results demonstrate a requirement for specific DNA sequences to trigger de novo DNA methylation, and repetitive elements as cis-regulatory factors to cooperate with advanced transcriptional repression in promoting methylation spreading.

Design, synthesis, pharmacological evaluation, and computational study of benzo[d] isothiazol-based small molecule inhibitors targeting PD-1/PD-l1 interaction.

Blockade of the programmed cell death-1 (PD-1)/programmed cell death ligand 1 (PD-L1) pathway is an attractive strategy for immunotherapy, but the clinical application of small molecule PD-1/PD-L1 inhibitors remains unclear. In this work, based on BMS-202 and our previous work YLW-106, a series of compounds with benzo[d]isothiazol structure as scaffold were designed and synthesized. Their inhibitory activity against PD-1/PD-L1 interaction was evaluated by a homogeneous time-resolved fluorescence (HTRF) assay. Among them, LLW-018 (27c) exhibited the most potent inhibitory activity with an IC50 value of 2.61 nM. The cellular level assays demonstrated that LLW-018 exhibited low cytotoxicity against Jurkat T and MDA-MB-231. Further cell-based PD-1/PD-L1 blockade bioassays based on PD-1 NFAT-Luc Jurkat cells and PD-L1 TCR Activator CHO cells indicated that LLW-018 could interrupt PD-1/PD-L1 interaction with an IC50 value of 0.88 µM. Multi-computational methods, including molecular docking, molecular dynamics, MM/GBSA, MM/PBSA, Metadynamics, and QM/MM MD were utilized on PD-L1 dimer complexes, which revealed the binding modes and dissociation process of LLW-018 and C2-symmetric small molecule inhibitor LCH1307. These results suggested that LLW-018 exhibited promising potency as a PD-1/PD-L1 inhibitor for further investigation.

Dietary Folate and Cofactors Accelerate Age-dependent p16 Epimutation to Promote Intestinal Tumorigenesis.

The extent to which non-genetic environmental factors, such as diet, contribute to carcinogenesis has been long debated. One potential mechanism for the effects of environmental factors is through epigenetic modifications that affect gene expression without changing the underlying DNA sequence. However, the functional cooperation between dietary factors and cancer-causing epigenetic regulation is largely unknown. Here, we use a mouse model of age-dependent p16 epimutation, in which the p16 gene activity is directly controlled by promoter DNA methylation. We show p16 epimutation is modulated by folate and cofactors in dietary supplementation, which leads to increased colon cancer risk. Importantly, our findings provide functional evidence concerning the safety of folate fortification in the general population. SIGNIFICANCE: Our study demonstrates that dietary folate and cofactors modulate tumor-suppressor gene methylation to increase intestinal tumorigenesis. Our findings highlight the need for monitoring the long-term safety of folate fortification in high-risk individuals.

Widespread and tissue specific age-related DNA methylation changes in mice.

Aberrant methylation of promoter CpG islands in cancer is associated with silencing of tumor-suppressor genes, and age-dependent hypermethylation in normal appearing mucosa may be a risk factor for human colon cancer. It is not known whether this age-related DNA methylation phenomenon is specific to human tissues. We performed comprehensive DNA methylation profiling of promoter regions in aging mouse intestine using methylated CpG island amplification in combination with microarray analysis. By comparing C57BL/6 mice at 3-mo-old versus 35-mo-old for 3627 detectable autosomal genes, we found 774 (21%) that showed increased methylation and 466 (13%) that showed decreased methylation. We used pyrosequencing to quantitatively validate the microarray data and confirmed linear age-related methylation changes for all 12 genomic regions examined. We then examined 11 changed genomic loci for age-related methylation in other tissues. Of these, three of 11 showed similar changes in lung, seven of 11 changed in liver, and six of 11 changed in spleen, though to a lower degree than the changes seen in colon. There was partial conservation between age-related hypermethylation in human and mouse intestines, and Polycomb targets in embryonic stem cells were enriched among the hypermethylated genes. Our findings demonstrate a surprisingly high rate of hyper- and hypomethylation as a function of age in normal mouse small intestine tissues and a strong tissue-specificity to the process. We conclude that epigenetic deregulation is a common feature of aging in mammals.

Genome architecture marked by retrotransposons modulates predisposition to DNA methylation in cancer.

Epigenetic silencing plays an important role in cancer development. An attractive hypothesis is that local DNA features may participate in differential predisposition to gene hypermethylation. We found that, compared with methylation-resistant genes, methylation-prone genes have a lower frequency of SINE and LINE retrotransposons near their transcription start site. In several large testing sets, this distribution was highly predictive of promoter methylation. Genome-wide analysis showed that 22% of human genes were predicted to be methylation-prone in cancer; these tended to be genes that are down-regulated in cancer and that function in developmental processes. Moreover, retrotransposon distribution marks a larger fraction of methylation-prone genes compared to Polycomb group protein (PcG) marking in embryonic stem cells; indeed, PcG marking and our predictive model based on retrotransposon frequency appear to be correlated but also complementary. In summary, our data indicate that retrotransposon elements, which are widespread in our genome, are strongly associated with gene promoter DNA methylation in cancer and may in fact play a role in influencing epigenetic regulation in normal and abnormal physiological states.

Season of conception in rural gambia affects DNA methylation at putative human metastable epialleles.

Throughout most of the mammalian genome, genetically regulated developmental programming establishes diverse yet predictable epigenetic states across differentiated cells and tissues. At metastable epialleles (MEs), conversely, epigenotype is established stochastically in the early embryo then maintained in differentiated lineages, resulting in dramatic and systemic interindividual variation in epigenetic regulation. In the mouse, maternal nutrition affects this process, with permanent phenotypic consequences for the offspring. MEs have not previously been identified in humans. Here, using an innovative 2-tissue parallel epigenomic screen, we identified putative MEs in the human genome. In autopsy samples, we showed that DNA methylation at these loci is highly correlated across tissues representing all 3 embryonic germ layer lineages. Monozygotic twin pairs exhibited substantial discordance in DNA methylation at these loci, suggesting that their epigenetic state is established stochastically. We then tested for persistent epigenetic effects of periconceptional nutrition in rural Gambians, who experience dramatic seasonal fluctuations in nutritional status. DNA methylation at MEs was elevated in individuals conceived during the nutritionally challenged rainy season, providing the first evidence of a permanent, systemic effect of periconceptional environment on human epigenotype. At MEs, epigenetic regulation in internal organs and tissues varies among individuals and can be deduced from peripheral blood DNA. MEs should therefore facilitate an improved understanding of the role of interindividual epigenetic variation in human disease.

Functional characterization of age-dependent p16 epimutation reveals biological drivers and therapeutic targets for colorectal cancer.

BACKGROUND: Methylation of the p16 promoter resulting in epigenetic gene silencing-known as p16 epimutation-is frequently found in human colorectal cancer and is also common in normal-appearing colonic mucosa of aging individuals. Thus, to improve clinical care of colorectal cancer (CRC) patients, we explored the role of age-related p16 epimutation in intestinal tumorigenesis. METHODS: We established a mouse model that replicates two common genetic and epigenetic events observed in human CRCs: Apc mutation and p16 epimutation. We conducted long-term survival and histological analysis of tumor development and progression. Colonic epithelial cells and tumors were collected from mice and analyzed by RNA sequencing (RNA-seq), quantitative PCR, and flow cytometry. We performed single-cell RNA sequencing (scRNA-seq) to characterize tumor-infiltrating immune cells throughout tumor progression. We tested whether anti-PD-L1 immunotherapy affects overall survival of tumor-bearing mice and whether inhibition of both epigenetic regulation and immune checkpoint is more efficacious. RESULTS: Mice carrying combined Apc mutation and p16 epimutation had significantly shortened survival and increased tumor growth compared to those with Apc mutation only. Intriguingly, colon tumors with p16 epimutation exhibited an activated interferon pathway, increased expression of programmed death-ligand 1 (Pdl1), and enhanced infiltration of immune cells. scRNA-seq further revealed the presence of Foxp3+ Tregs and γδT17 cells, which contribute to an immunosuppressive tumor microenvironment (TME). Furthermore, we showed that a combined therapy using an inhibitor of DNA methylation and a PD-L1 immune checkpoint inhibitor is more effective for improving survival in tumor-bearing mice than blockade of either pathway alone. CONCLUSIONS: Our study demonstrated that age-dependent p16 epimutation creates a permissive microenvironment for malignant transformation of polyps to colon cancer. Our findings provide a mechanistic rationale for future targeted therapy in patients with p16 epimutation.

Epigenetic maturation in colonic mucosa continues beyond infancy in mice.

Monozygotic twin and other epidemiologic studies indicate that epigenetic processes may play an important role in the pathogenesis of inflammatory bowel diseases that commonly affect the colonic mucosa. The peak onset of these disorders in young adulthood suggests that epigenetic changes normally occurring in the colonic mucosa shortly before adulthood could be important etiologic factors. We assessed developmental changes in colitis susceptibility during the physiologically relevant period of childhood in mice [postnatal day 30 (P30) to P90] and concurrent changes in DNA methylation and gene expression in murine colonic mucosa. Susceptibility to colitis was tested in C57BL/6J mice with the dextran sulfate sodium colitis model. Methylation specific amplification microarray (MSAM) was used to screen for changes in DNA methylation, with validation by bisulfite pyrosequencing. Gene expression changes were analyzed by microarray expression profiling and real time RT-PCR. Mice were more susceptible to chemically induced colitis at P90 than at P30. DNA methylation changes, however, were not extensive; of 23 743 genomic intervals interrogated, only 271 underwent significant methylation alteration during this developmental period. We found an excellent correlation between the MSAM and bisulfite pyrosequencing at 11 gene associated intervals validated (R(2) = 0.89). Importantly, at the genes encoding galectin-1 (Lgals1), and mothers against decapentaplegic homolog 3 or Smad3, both previously implicated in murine colitis, developmental changes in DNA methylation from P30 to P90 were inversely correlated with expression. Colonic mucosal epigenetic maturation continues through early adulthood in the mouse, and may contribute to the age-associated increase in colitis susceptibility. Transcript Profiling: Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/), accession numbers: GSE18031 (DNA methylation arrays), GSE19506 (gene expression arrays).

Foxa1 functions as a pioneer transcription factor at transposable elements to activate Afp during differentiation of embryonic stem cells.

Epigenetic control of genes that are silent in embryonic stem cells, but destined for expression during differentiation, includes distinctive hallmarks, such as simultaneous activating/repressing (bivalent) modifications of chromatin and DNA hypomethylation at enhancers of gene expression. Although alpha-fetoprotein (Afp) falls into this class of genes, as it is silent in pluripotent stem cells and activated during differentiation of endoderm, we find that Afp chromatin lacks bivalent histone modifications. However, critical regulatory sites for Afp activation, overlapping Foxa1/p53/Smad-binding elements, are located within a 300-bp region lacking DNA methylation, due to transposed elements underrepresented in CpG sequences: a short interspersed transposable element and a medium reiterated sequence 1 element. Forkhead family member Foxa1 is activated by retinoic acid treatment of embryonic stem cells, binds its DNA consensus site within the short interspersed transposable/medium reiterated sequence 1 elements, and displaces linker histone H1 from silent Afp chromatin. Small interfering RNA depletion of Foxa1 showed that Foxa1 is essential in providing chromatin access to transforming growth factor beta-activated Smad2 and Smad4 and their subsequent DNA binding. Together these transcription factors establish highly acetylated chromatin and promote expression of Afp. Foxa1 acts as a pioneer transcription factor in de novo activation of Afp, by exploiting a lack of methylation at juxtaposed transposed elements, to bind and poise chromatin for intersection with transforming growth factor beta signaling during differentiation of embryonic stem cells.

Epigenomic profiling indicates a role for DNA methylation in early postnatal liver development.

The question of whether DNA methylation contributes to the stabilization of gene expression patterns in differentiated mammalian tissues remains controversial. Using genome-wide methylation profiling, we screened 3757 gene promoters for changes in methylation during postnatal liver development to test the hypothesis that developmental changes in methylation and expression are temporally correlated. We identified 31 genes that gained methylation and 111 that lost methylation from embryonic day 17.5 to postnatal day 21. Promoters undergoing methylation changes in postnatal liver tended not to be associated with CpG islands. At most genes studied, developmental changes in promoter methylation were associated with expression changes, suggesting both that transcriptional inactivity attracts de novo methylation, and that transcriptional activity can override DNA methylation and successively induce developmental hypomethylation. These in vivo data clearly indicate a role for DNA methylation in mammalian differentiation, and provide the novel insight that critical windows in mammalian developmental epigenetics extend well beyond early embryonic development.