Dynamics of activating and repressive histone modifications in Drosophila neural stem cell lineages and brain tumors.

During central nervous system development, spatiotemporal gene expression programs mediate specific lineage decisions to generate neuronal and glial cell types from neural stem cells (NSCs). However, little is known about the epigenetic landscape underlying these highly complex developmental events. Here, we perform ChIP-seq on distinct subtypes of Drosophila FACS-purified NSCs and their differentiated progeny to dissect the epigenetic changes accompanying the major lineage decisions in vivo By analyzing active and repressive histone modifications, we show that stem cell identity genes are silenced during differentiation by loss of their activating marks and not via repressive histone modifications. Our analysis also uncovers a new set of genes specifically required for altering lineage patterns in type II neuroblasts (NBs), one of the two main Drosophila NSC identities. Finally, we demonstrate that this subtype specification in NBs, unlike NSC differentiation, requires Polycomb-group-mediated repression.

A comparative study of ChIP-seq sequencing library preparation methods.

BACKGROUND: ChIP-seq is the primary technique used to investigate genome-wide protein-DNA interactions. As part of this procedure, immunoprecipitated DNA must undergo "library preparation" to enable subsequent high-throughput sequencing. To facilitate the analysis of biopsy samples and rare cell populations, there has been a recent proliferation of methods allowing sequencing library preparation from low-input DNA amounts. However, little information exists on the relative merits, performance, comparability and biases inherent to these procedures. Notably, recently developed single-cell ChIP procedures employing microfluidics must also employ library preparation reagents to allow downstream sequencing. RESULTS: In this study, seven methods designed for low-input DNA/ChIP-seq sample preparation (Accel-NGS® 2S, Bowman-method, HTML-PCR, SeqPlex™, DNA SMART™, TELP and ThruPLEX®) were performed on five replicates of 1 ng and 0.1 ng input H3K4me3 ChIP material, and compared to a "gold standard" reference PCR-free dataset. The performance of each method was examined for the prevalence of unmappable reads, amplification-derived duplicate reads, reproducibility, and for the sensitivity and specificity of peak calling. CONCLUSIONS: We identified consistent high performance in a subset of the tested reagents, which should aid researchers in choosing the most appropriate reagents for their studies. Furthermore, we expect this work to drive future advances by identifying and encouraging use of the most promising methods and reagents. The results may also aid judgements on how comparable are existing datasets that have been prepared with different sample library preparation reagents.

The tumor suppressors Brat and Numb regulate transit-amplifying neuroblast lineages in Drosophila.

In both vertebrates and insects, neurons typically arise from neural stem cells or terminally dividing intermediate progenitors. Here, we describe another mode of neurogenesis where neural stem cells generate secondary precursors that undergo multiple rounds of self-renewing transit-amplifying divisions. We identify the Posterior Asense-Negative (PAN) neuroblasts, which do not express the transcription factors Asense or Prospero. PAN neuroblasts rely on the segregating determinants Numb and Brat to generate smaller, secondary neuroblasts that in turn give rise to ganglion mother cells (GMCs) and neurons throughout larval development. In brat or numb mutants, misspecified secondary neuroblasts are unable to produce differentiated progeny and initiate tumor-like overgrowth. In prospero mutants, however, tumors arise from GMCs while secondary neuroblasts are correctly specified. Our data describe a transit-amplifying lineage in the Drosophila nervous system and suggest that different vulnerabilities in intermediate cell types can affect the outcome of tumor suppressor loss in stem cell lineages.

The Drosophila NuMA Homolog Mud regulates spindle orientation in asymmetric cell division.

During asymmetric cell division, the mitotic spindle must be properly oriented to ensure the asymmetric segregation of cell fate determinants into only one of the two daughter cells. In Drosophila neuroblasts, spindle orientation requires heterotrimeric G proteins and the G alpha binding partner Pins, but how the Pins-G alphai complex interacts with the mitotic spindle is unclear. Here, we show that Pins binds directly to the microtubule binding protein Mud, the Drosophila homolog of NuMA. Like NuMA, Mud can bind to microtubules and enhance microtubule polymerization. In the absence of Mud, mitotic spindles in Drosophila neuroblasts fail to align with the polarity axis. This can lead to symmetric segregation of the cell fate determinants Brat and Prospero, resulting in the mis-specification of daughter cell fates and tumor-like over proliferation in the Drosophila nervous system. Our data suggest a model in which asymmetrically localized Pins-G alphai complexes regulate spindle orientation by directly binding to Mud.