LSH, a member of the SNF2 family of chromatin remodeling ATPases encoded by the Hells gene, is essential for normal levels of DNA methylation in the mammalian genome. While the role of LSH in the methylation of repetitive DNA sequences is well characterized, its contribution to the regulation of DNA methylation and the expression of proteincoding genes has not been studied in detail. In this report we investigate genome-wide patterns of DNA methylation at gene promoters in Hells -/-mouse embryonic fibroblasts (MEFs). We find that in the absence of LSH, DNA methylation is lost or significantly reduced at~20% of all normally methylated promoter sequences. As a consequence, a large number of genes are misexpressed in Hells -/-MEFs. Comparison of Hells -/-MEFs with wild-type MEFs and embryonic stem (ES) cells suggests that LSH is important for de novo DNA methylation events that accompany the establishment and differentiation of embryonic lineage cells. We further show that the generation of normal DNA methylation patterns and stable gene silencing at specific promoters require cooperation between LSH and the G9a/GLP complex of histone methylases. At such loci, G9a recruitment is compromised when LSH is absent or greatly reduced. Taken together, our data suggest a mechanism whereby LSH promotes binding of DNA methyltransferases and the G9a/GLP complex to specific loci and facilitates developmentally programmed DNA methylation and stable gene silencing during lineage commitment and differentiation.

BackgroundAdvances in sequencing technologies and bioinformatics have made the analysis of microbial communities almost routine. Nonetheless, the need remains to improve on the techniques used for gathering such data, including increasing throughput while lowering cost and benchmarking the techniques so that potential sources of bias can be better characterized.MethodsWe present a triple-index amplicon sequencing strategy to sequence large numbers of samples at significantly lower c ost and in a shorter timeframe compared to existing methods. The design employs a two-stage PCR protocol, incorpo rating three barcodes to each sample, with the possibility to add a fourth-index. It also includes heterogeneity spacers to overcome low complexity issues faced when sequencing amplicons on Illumina platforms.ResultsThe library preparation method was extensively benchmarked through analysis of a mock community in order to assess biases introduced by sample indexing, number of PCR cycles, and template concentration. We further evaluated the method through re-sequencing of a standardized environmental sample. Finally, we evaluated our protocol on a set of fecal samples from a small cohort of healthy adults, demonstrating good performance in a realistic experimental setting. Between-sample variation was mainly related to batch effects, such as DNA extraction, while sample indexing was also a significant source of bias. PCR cycle number strongly influenced chimera formation and affected relative abundance estimates of species with high GC content. Libraries were sequenced using the Illumina HiSeq and MiSeq platforms to demonstrate that this protocol is highly scalable to sequence thousands of samples at a very low cost.ConclusionsHere, we provide the most comprehensive study of performance and bias inherent to a 16S rRNA gene amplicon sequencing method to date. Triple-indexing greatly reduces the number of long custom DNA oligos required for library preparation, while the inclusion of variable length heterogeneity spacers minimizes the need for PhiX spike-in. This design results in a significant cost reduction of highly multiplexed amplicon sequencing. The biases we characterize highlight the need for highly standardized protocols. Reassuringly, we find that the biological signal is a far stronger structuring factor than the various sources of bias.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-017-0279-1) contains supplementary material, which is available to authorized users.

The transcription factor c-Myb is involved in early differentiation and proliferation of haematopoietic cells, where it operates as a regulator of self-renewal and multi-lineage differentiation. Deregulated c-Myb plays critical roles in leukaemias and other human cancers. Due to its role as a master regulator, we hypothesized it might function as a pioneer transcription factor. Our approach to test this was to analyse a mutant of c-Myb, D152V, previously reported to cause haematopoietic defects in mice by an unknown mechanism. Our transcriptome data from K562 cells indicates that this mutation specifically affects c-Myb's ability to regulate genes involved in differentiation, causing failure in c-Myb's ability to block differentiation. Furthermore, we see a major effect of this mutation in assays where chromatin opening is involved. We show that each repeat in the minimal DNA-binding domain of c-Myb binds to histones and that D152V disrupts histone binding of the third repeat. ATAC-seq data indicates this mutation impairs the ability of c-Myb to cause chromatin opening at specific sites. Taken together, our findings support that c-Myb acts as a pioneer factor and show that D152V impairs this function. The D152V mutant is the first mutant of a transcription factor specifically destroying pioneer factor function.