Epigenomic Profiling of Human CD4+ T Cells Supports a Linear Differentiation Model and Highlights Molecular Regulators of Memory Development.

The impact of epigenetics on the differentiation of memory T (Tmem) cells is poorly defined. We generated deep epigenomes comprising genome-wide profiles of DNA methylation, histone modifications, DNA accessibility, and coding and non-coding RNA expression in naive, central-, effector-, and terminally differentiated CD45RA+ CD4+ Tmem cells from blood and CD69+ Tmem cells from bone marrow (BM-Tmem). We observed a progressive and proliferation-associated global loss of DNA methylation in heterochromatic parts of the genome during Tmem cell differentiation. Furthermore, distinct gradually changing signatures in the epigenome and the transcriptome supported a linear model of memory development in circulating T cells, while tissue-resident BM-Tmem branched off with a unique epigenetic profile. Integrative analyses identified candidate master regulators of Tmem cell differentiation, including the transcription factor FOXP1. This study highlights the importance of epigenomic changes for Tmem cell biology and demonstrates the value of epigenetic data for the identification of lineage regulators.

Combining transcription factor binding affinities with open-chromatin data for accurate gene expression prediction.

The binding and contribution of transcription factors (TF) to cell specific gene expression is often deduced from open-chromatin measurements to avoid costly TF ChIP-seq assays. Thus, it is important to develop computational methods for accurate TF binding prediction in open-chromatin regions (OCRs). Here, we report a novel segmentation-based method, TEPIC, to predict TF binding by combining sets of OCRs with position weight matrices. TEPIC can be applied to various open-chromatin data, e.g. DNaseI-seq and NOMe-seq. Additionally, Histone-Marks (HMs) can be used to identify candidate TF binding sites. TEPIC computes TF affinities and uses open-chromatin/HM signal intensity as quantitative measures of TF binding strength. Using machine learning, we find low affinity binding sites to improve our ability to explain gene expression variability compared to the standard presence/absence classification of binding sites. Further, we show that both footprints and peaks capture essential TF binding events and lead to a good prediction performance. In our application, gene-based scores computed by TEPIC with one open-chromatin assay nearly reach the quality of several TF ChIP-seq data sets. Finally, these scores correctly predict known transcriptional regulators as illustrated by the application to novel DNaseI-seq and NOMe-seq data for primary human hepatocytes and CD4+ T-cells, respectively.

An analysis of PIK3CA hotspot mutations and response to neoadjuvant therapy in breast cancer patients from four prospective clinical trials.

PURPOSE: The PI3K signaling pathway is frequently dysregulated in breast cancer (BC), and mutations in PIK3CA, are relevant for therapy resistance in HER2pos BC. Mutations in exons 9 or exon 20 may have different impact on response to neoadjuvant chemotherapy-based treatment regimens. EXPERIMENTAL DESIGN: We investigated PIK3CA mutations in 1691 early BC patients, randomized in four neoadjuvant multicenter trials: GeparQuattro (NCT00288002), GeparQuinto (NCT00567554), GeparSixto (NCT01426880) and GeparSepto (NCT01583426). The role of different PIK3CA exons and hotspots for pathological complete response (pCR) after neoadjuvant chemotherapy (NACT) and patient survival was evaluated for distinct molecular subgroups and anti-HER2 treatment procedures. RESULTS: A total of 302 patients (17.9%) of the full cohort of 1691 patients had a tumor with a PIK3CA mutation, with a different prevalence in molecular subgroups: luminal/HER2neg 95 of 404 patients (23.5%), HER2pos 170 of 819 patients (20.8%) and TNBC 37 of 431 patients (7.9%). We identified mutations in PIK3CA exon 20 to be linked with worse response to anti-HER2 treatment (OR=0.507, 95%CI 0.320-0.802, p=0.004), especially in HR positive HER2 positive BC (OR=0.445, 95%CI 0.237-0.837, p=0.012). In contrast, exon 9 hotspot mutations p.E452K and p.E545K revealed no noteworthy differences in response therapy response. Luminal/HER2neg patients show a trend to have worse treatment response when PIK3CA was mutated. Interestingly, patients with residual disease after neoadjuvant treatment, have better survival when PIK3CA was mutated. CONCLUSIONS: PIK3CA hotspot mutation p.H1047R are associated with worse pCR rates after NACT in HER2pos BC, while hotspot mutations in exon 9 seems to have less impact.

FARP2, HDLBP and PASK are downregulated in a patient with autism and 2q37.3 deletion syndrome.

We describe a patient with autism and brachymetaphalangy, meeting criteria for 2q37 deletion syndrome (also called Albright Hereditary Osteodystrophy-like syndrome or Brachydactyly-Mental Retardation syndrome, OMIM 600430). Our molecular cytogenetic studies, including array comparative genomic hybridization (aCGH) and fluorescence in situ hybridization (FISH), define the extent of the de novo deletion to a 3.5 Mb region on 2q37.3. Although a number of reports of patients with 2q37 deletion syndrome have been published, it remains unclear if gene expression and/or translation are altered by the deletion, thus contributing to the observed phenotypes. To address this question, we selected several candidate genes for the neuropsychiatric and skeletal anomalies found in this patient (autism and brachymetaphalangy). The deleted region in 2q37.3 includes the FERM, RhoGEF and pleckstrin domain protein 2 (FARP2), glypican 1 (GPC1), vigilin (HDLBP), kinesin family member 1A (KIF1A) and proline-alanine-rich STE20-related kinase (PASK), all of which are involved in skeletal or neural differentiation processes. Expression analyses of these genes were performed using RNA from lymphoblastoid cell lines of the patient and his family members. Here we demonstrate that three of these genes, FARP2, HDLBP, and PASK, are considerably downregulated in the patient's cell line. We hypothesize that haploinsufficiency of these genes may have contributed to the patient's clinical phenotype.

Integrative analysis of single-cell expression data reveals distinct regulatory states in bidirectional promoters.

BACKGROUND: Bidirectional promoters (BPs) are prevalent in eukaryotic genomes. However, it is poorly understood how the cell integrates different epigenomic information, such as transcription factor (TF) binding and chromatin marks, to drive gene expression at BPs. Single-cell sequencing technologies are revolutionizing the field of genome biology. Therefore, this study focuses on the integration of single-cell RNA-seq data with bulk ChIP-seq and other epigenetics data, for which single-cell technologies are not yet established, in the context of BPs. RESULTS: We performed integrative analyses of novel human single-cell RNA-seq (scRNA-seq) data with bulk ChIP-seq and other epigenetics data. scRNA-seq data revealed distinct transcription states of BPs that were previously not recognized. We find associations between these transcription states to distinct patterns in structural gene features, DNA accessibility, histone modification, DNA methylation and TF binding profiles. CONCLUSIONS: Our results suggest that a complex interplay of all of these elements is required to achieve BP-specific transcriptional output in this specialized promoter configuration. Further, our study implies that novel statistical methods can be developed to deconvolute masked subpopulations of cells measured with different bulk epigenomic assays using scRNA-seq data.

Epigenetic dynamics of monocyte-to-macrophage differentiation.

BACKGROUND: Monocyte-to-macrophage differentiation involves major biochemical and structural changes. In order to elucidate the role of gene regulatory changes during this process, we used high-throughput sequencing to analyze the complete transcriptome and epigenome of human monocytes that were differentiated in vitro by addition of colony-stimulating factor 1 in serum-free medium. RESULTS: Numerous mRNAs and miRNAs were significantly up- or down-regulated. More than 100 discrete DNA regions, most often far away from transcription start sites, were rapidly demethylated by the ten eleven translocation enzymes, became nucleosome-free and gained histone marks indicative of active enhancers. These regions were unique for macrophages and associated with genes involved in the regulation of the actin cytoskeleton, phagocytosis and innate immune response. CONCLUSIONS: In summary, we have discovered a phagocytic gene network that is repressed by DNA methylation in monocytes and rapidly de-repressed after the onset of macrophage differentiation.

Evaluation of BMP4 and its specific inhibitor NOG as candidates in human neural tube defects (NTDs).

Neural tube defects (NTD) are among the most common congenital malformations in humans. The current view is that there are no major genes causing NTDs, but combinations of sequence variants in different genes have additive effects on determining the malformation. Therefore it is important to identify such sequence variants to get a better understanding of NTD pathogenesis. Studies on animal models have shown that BMP4 and NOG are involved in the patterning of the neural tube. We therefore performed a single-strand conformation analysis (SSCA) mutation screen for both genes in 179 spina bifida aperta (SBA) patients. Our SSCA screen revealed four missense mutations in BMP4 and one in NOG. It is likely that these mutations have acted together with other gene variants in independently segregating loci as susceptibility factors in these SBA cases. In addition, a case-control association study provides evidence for a genotype disequilibrium of BMP4 polymorphism 455T-->C (V152A) in exon 5. The frequency of the heterozygous 455TC genotype is lower in cases than in controls (nominal P=0.017), although allele frequencies are similar in both groups. A possible explanation for this finding might be that BMP4 455TC heterozygosity at this site is a protective factor in the normal population, although this hypothesis cannot be proven to date.