Gene expression regulation by the Chromodomain helicase DNA-binding protein 9 (CHD9) chromatin remodeler is dispensable for murine development

Abstract

Chromodomain helicase DNA-binding (CHD) chromatin remodelers regulate transcription and DNA repair. They govern cell-fate decisions during embryonic development and are often deregulated in human pathologies. Chd1-8 show upon germline disruption pronounced, often developmental lethal phenotypes. Here we show that contrary to Chd1-8 disruption, Chd9-/-animals are viable, fertile and display no developmental defects or disease predisposition. Germline deletion of Chd9 only moderately affects gene expression in tissues and derived cells, whereas acute depletion in human cancer cells elicits more robust changes suggesting that CHD9 is a highly context-dependent chromatin regulator that, surprisingly, is dispensable for mouse development.

Fig 1. Generation of the Chd9 knockout results in complete removal of the locus coding potential.

(A) (Above) Representation of the CHD9 protein domains- CD (chromodomain), BRK (Brahma-Kismet domain), and (bellow) targeting strategy whereby Cre–mediated deletion removed coding potential of the locus. (B) Left panel, Southern blot analysis of genomic DNA from adult organs and ES cells from wild type and Chd9 knockout animals, using external and internal radioactive probes. Right panel, PCR detection of deleted allele using primers 117kb apart. (C) Left panel, Northern blot mRNA detection in adult brain and ES cells using 3’UTR and internal cDNA PCR–probes. Right panel, Western blot analysis showing complete depletion of the CHD9 protein in ES cells. Vinculin used as a loading control.

Fig 2. Chd9 deletion does not affect murine survival and has moderate effect on gene expression.

(A) Chd9 knockout animals are viable, fertile and display no overt phenotypes. Kaplan–Meier survival curves reveal comparable life expectancies of wild-type, heterozygous and homozygous knockout animals (ns = not significant, log-rank test p–value = 0.272). (B) Venn diagram representation of the overlap between differentially expressed genes (DEGs) identified in Chd9^–/–MEFs, ESCs, E15 brain and liver. Numbers in parenthesis represent total number of DEGs between Chd9^–/–and Chd9^+/+ cells and organs (p-adj < 0.05, log₂FC (fold change) > ±1). The numbers in black indicate the number of non-overlapping genes between analyzed groups.

Fig 3. Chd9 knockout animals display mild effect on hematopoietic stem cells.

(A,B) Flow cytometric analysis of Chd9 wild-type (WT, n = 3) and knockout (KO, n = 4) bone marrow (BM) stem cell and progenitor cells revealed expansion of short–term hematopoietic stem cells (red asterisk denotes p–value: 0.018, ST–HSC). Other BM hematopoietic stem cell and progenitor populations display no statistically significant differences. Data plotted as frequency of hematopoietic cells, mean ±SD, p-value calculated using two-tailed Student’s t–test. HSCP- hematopoietic stem cells and progenitors: Lin−(lineage negative), HSC (hematopoietic stem cells), MPP (multipotent progenitor cells), LT-HSC (long-term hematopoietic stem cells). Progenitors: IgM⁺ (B-cell progenitors), CMP (common myeloid precursor), GMP (granulocyte-monocyte progenitors), MEP (megakaryocyte-erythroid progenitors).

Fig 4. Chd9 deletion does not affect latency or gene expression of EμMyc–driven lymphomagenesis.

(A) Kaplan–Meier plot shows comparable tumor-free survival of EμMyc;Chd9 animals (ns = not significant, log-rank test, p–value = 0.78). (B) Venn diagram representation of the overlap between differentially expressed genes (DEGs) identified in EμMyc;Chd9^+/–(left) and EμMyc;Chd9^–/–(right) compared to control EμMyc;Chd9^+/+ lymphomas. Numbers in parenthesis represent total number of DEGs (p-adj < 0.05, log₂FC (fold change) > ±1). The DEGs common for EμMyc;Chd9^+/–and EμMyc;Chd9^+/+ samples are highlighted in black and non-overlapping genes in red.

Fig 5. CHD9 depletion deregulates gene expression in human SNB19 and K562 cancer cells.

(A, B) Western blots reveal depletion of the CHD9 protein by RNA-interference 48-hrs post doxycycline addition (±DOX). (C, D) Heatmaps of the top 20 most significant DEGs based on the p-adj value in SNB19 and K562 cells upon CHD9 knockdown. The color scale is based on the normalized read count values.