Unknown,Transcriptomics,Genomics,Proteomics

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Chx10 consolidates V2a interneuron identity through two distinct gene repression modes [RNA-Seq]

ABSTRACT: During development, two cell-types born from closely related progenitor pools often express the identical transcriptional regulators despite their completely distinct characteristics. This phenomenon highlights the necessity of the mechanism that operates to segregate the identities of the two cell-types throughout differentiation after initial fate commitment. To understand this mechanism, we investigated the fate specification of spinal V2a interneurons, which share important developmental genes with motor neurons (MNs). Here we demonstrate that the paired homeodomain factor Chx10 functions as a critical determinant for V2a fate and is required to consolidate V2a identity in postmitotic neurons. Chx10 actively promotes V2a fate, downstream of the LIM-homeodomain factor Lhx3, while concomitantly suppressing MN developmental program by preventing the MN-specific transcription complex from binding and activating MN genes. This dual activity enables Chx10 to effectively separate V2a and MN pathways. Together, our study uncovers a widely applicable gene regulatory principle for segregating related cell fates. RNA samples from Chx10-ESC-derived MNs were prepared for sequencing according to the Illumina protocol, and sequenced on the Illumina HiSeq 2000. We will then compare the transcriptome changes between -Dox (no Chx10) and +Dox (Chx10) in order to identify genes rregulated by Chx10.

ORGANISM(S): Mus musculus

SUBMITTER: Jae Lee

PROVIDER: E-GEOD-83873 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The motor neuron (MN)–hexamer complex consisting of LIM homeobox 3, Islet-1, and nuclear LIM interactor is a key determinant of motor neuron specification and differentiation. To gain insights into the transcriptional network in motor neuron development, we performed a genome-wide ChIP-sequencing analysis and found that the MN–hexamer directly regulates a wide array of motor neuron genes by binding to the HxRE (hexamer response element) shared among the target genes. Interestingly, STAT3-binding motif is highly enriched in the MN–hexamer–bound peaks in addition to the HxRE. We also found that a transcriptionally active form of STAT3 is expressed in embryonic motor neurons and that STAT3 associates with the MN–hexamer, enhancing the transcriptional activity of the MN–hexamer in an upstream signal-dependent manner. Correspondingly, STAT3 was needed for motor neuron differentiation in the developing spinal cord. Together, our studies uncover crucial gene regulatory mechanisms that couple MN–hexamer and STAT-activating extracellular signals to promote motor neuron differentiation in vertebrate spinal cord. To explain our experimental scheme briefly, we are interested in finding target sites for the dimer of transcription factors Isl1 and Lhx3. To mimic the biological activity of Isl1/Lhx3 dimer, we made Isl1-Lhx3 fusion and found that Isl1-Lhx3 has a potent biological activity in multiple systems (i.e. generation of ectopic motor neurons). Then we made ES cell line that induces Flag-tagged Isl1-Lhx3 expression upon Dox treatment. These *mouse* ES cells differentiate to motor neurons (iMN-ESCs) when treated with Dox following EB formation. To identify genomic binding sites of Isl1-Lhx3 (Flag-tagged), we performed ChIP with Flag antibody (pull down of Flag-Isl1-Lhx3) in ES cells treated with Dox. ChIP with Flag antibody in ES cells treated with vehicle (no Dox) was done as a negative control in parallel, and sequenced along with +Dox sample. We have done these experiments twice (two sets).

Project description:Motor neuron (MN), together with interneuron, are the two major neuronal types in spinal cord. MNs control muscle movement and are essential for breathing, walking and fine motor skills. Malfunction of MNs is frequently associated with neuronal diseases such as spinal muscular atrophy and amyotrophic lateral sclerosis. To establish normal function, MNs need to be differentiated properly from neural progenitor cells. Thus, it is of great importance to study the mechanism for MN specification. This study focuses on Mnx1 - a conserved homeobox transcription factor gets expressed during MN specification. Mnx1 is also the most widely used MN marker. Previous studies reported that Mnx1 mutation in mouse causes early lethality - probably by affecting the controlling over respiratory system. However, the exact role of Mnx1 for MN identity remains poorly understood. Taking advantage of a recently developed in vitro model for efficient MN induction from mouse ES cells, we combined experimental and OMICs techniques to systematically investigate the molecular function of Mnx1. We identified thousands of Mnx1 binding sites - many are co-bound by core MN factor Lhx3 and Isl1 and lack active histone marks. Disruption of Mnx1 causes increased expression of 85 genes - while only 14 genes get down-regulated. Up-regulated genes are enriched with neuronal functions, usually get expressed during MN differentiation, and tend to have higher expression in motor neurons and brain compared with other tissues – indicating Mnx1 may fine tune neuronal genes to desired level. However, only a dozen of regions with increased H3K27ac marks are bound by Mnx1, and only two up-regulated genes (Pbx3 and Pou6f2) are identified as putative direct targets of Mnx1 - both are core neuronal factors with the potential to regulate other differential neuronal genes. These results suggest that Mnx1 may contribute to MN identity by fine-tuning the expression of many neuronal genes through direct regulation of a few core neuronal transcription factors. In summary, this study represents the first systematic investigation about the molecular function of the core MN factor Mnx1. It clarifies the mechanism of Mnx1 for MN identity, and also improves the understanding about the regulation mode of homeobox transcription factors.

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Chx10 consolidates V2a interneuron identity through two distinct gene repression modes [RNA-Seq]

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