Dataset Information

The hematopoietic master transcription factor PU.1 requires its interaction with the SWI/SNF remodeler to access chromatin de novo

ABSTRACT: PU.1 is a prototype master transcription factor (TF) of hematopoietic cell differentiation with diverse roles in different lineages. Analysis of its genome-wide binding pattern across PU.1 expressing cell types revealed manifold cell type-specific binding patterns. They are not consistent with the epigenetic and chromatin constraints to PU.1 binding observed in vitro, suggesting that PU.1 requires auxiliary factors to access DNA in vivo. Using a model of transient mRNA expression we show that PU.1 induction leads to the extensive remodeling of chromatin, redistribution of partner transcription factors and rapid initiation of a myeloid gene expression program in heterologous cell types. By probing PU.1 mutants for defects in chromatin access and screening for PU.1 proximal proteins in vivo, we found that its N-terminal acidic domain was required for the recruitment of SWI/SNF remodeling complexes, de novo chromatin access and stable binding as well as the redistribution of partner TFs.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Cell, Cell Suspension Culture

SUBMITTER: Andreas Schmidt

LAB HEAD: Axel Imhof

PROVIDER: PXD013167 | Pride | 2019-11-20

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20180209_QEX1_AS_JM_BioID_01_THP1_PU1_BirA1.raw	Raw
	20180209_QEX1_AS_JM_BioID_03_THP1_NLS_BirA1.raw	Raw
	20180209_QEX1_AS_JM_BioID_04_THP1_PU1_BirA2.raw	Raw
	20180209_QEX1_AS_JM_BioID_06_THP1_NLS_BirA2.raw	Raw
	20180209_QEX1_AS_JM_BioID_07_THP1_PU1_BirA3.raw	Raw

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Publications

Mechanisms governing the pioneering and redistribution capabilities of the non-classical pioneer PU.1.

Minderjahn Julia J Schmidt Andreas A Fuchs Andreas A Schill Rudolf R Raithel Johanna J Babina Magda M Schmidl Christian C Gebhard Claudia C Schmidhofer Sandra S Mendes Karina K Ratermann Anna A Glatz Dagmar D Nützel Margit M Edinger Matthias M Hoffmann Petra P Spang Rainer R Längst Gernot G Imhof Axel A Rehli Michael M

Nature communications 20200121 1

Establishing gene regulatory networks during differentiation or reprogramming requires master or pioneer transcription factors (TFs) such as PU.1, a prototype master TF of hematopoietic lineage differentiation. To systematically determine molecular features that control its activity, here we analyze DNA-binding in vitro and genome-wide in vivo across different cell types with native or ectopic PU.1 expression. Although PU.1, in contrast to classical pioneer factors, is unable to access nucleosom ...[more]

PMID: 31964861

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Project description:In mammalian cells transcription factors (TFs) preferentially bind sites contained in regions of high nucleosomal occupancy, as determined by nucleotide-dependent computational analysis. This observation suggests that nucleosomes may act as gatekeepers of TF binding sites. We hypothesized that in mammalian genomes the information controlling nucleosome assembly may partially coincide with the information that enables TFs to recognize cognate sites in cis-regulatory elements while ignoring the myriad of non-functional, randomly occurring consensus binding sites. This way, nucleosome-mediated masking would be coupled to TF binding site functionality. The hematopoietic master regulator Pu.1 maintained nucleosome depletion at macrophage-specific enhancers that were otherwise occupied by nucleosomes in other cell types and in reconstituted chromatin. We identified a minimal set of DNA sequence and shape features that predicted Pu.1 binding with 78% accuracy. The same features predicted nucleosome occupancy in cells where Pu.1 was not expressed with higher accuracy than specifically designed models. Control of nucleosome deposition by DNA sequence and shape features that also specify TF consensus site functionality may allow maintaining the gatekeeper function of nucleosomes during evolution of cis-regulatory elements. Chromatin immuno-precipitations of the transcription factor Pu.1 followed by multiparallel sequencing performed in murine bone marrow-derived macrophages. Experiments carried out in cells infected either with a retroviral vector containing a short hairpin targeting Pu.1 or with the empty vector as control. The shPU.1 hairpin (sequence available upon request) was selected among five designed using a publicly available software (http://katahdin.mssm.edu/siRNA) and was cloned in a modified version of TtRMPVIR inducible retroviral vector (Genbank HQ456318) in which the puromycin resistance gene was inserted. The empty vector, containing an sh-Renilla sequence, was used as control. Chromatin immuno-precipitations of the transcription factor PU.1 binding to in vitro reconstituted chromatin followed by multiparallel sequencing Micrococcal nuclease digestion of chromatin extracted from bone marrow derived macrophages followed by multiparallel sequencing . Experiments were carried out in untreated cells (4 replicates) and cells infected either with a retroviral vector containing a short hairpin targeting Pu.1 or with the empty vector as control (2 replicates). The shPU.1 hairpin (sequence available upon request) was selected among five designed using a publicly available software (http://katahdin.mssm.edu/siRNA) and was cloned in a modified version of TtRMPVIR inducible retroviral vector (Genbank HQ456318) in which the puromycin resistance gene was inserted. The empty vector, containing an sh-Renilla sequence, was used as control. Micrococcal nuclease digestion of in vitro reconstituted chromatin followed by multiparallel sequencing