Characterization of the CHD family of proteins

Abstract

The murine gene CHD1 (MmCHD1) was previously isolated in a search for proteins that bound a DNA promoter element. The presence of chromo (chromatin organization modifier) domains and an SNF2-related helicase/ATPase domain led to speculation that this gene regulated chromatin structure or gene transcription. This study describes the cloning and characterization of three novel human genes related to MmCHD1. Examination of sequence databases produced several more related genes, most of which were not known to be similar to MmCHD1, yielding a total of 12 highly conserved CHD genes from organisms as diverse as yeast and mammals. The major region of sequence variation is in the C-terminal part of the protein, a region with DNA-binding activity in MmCHD1. Targeted deletion of ScCHD1, the sole Saccharomyces cerevesiae CHD gene, was performed with deletion strains being less sensitive than wild type to the cytotoxic effect of 6-azauracil. This finding suggested that enhanced transcriptional arrest at RNA polymerase II pause sites due to 6-azauracil-induced nucleotide pool depletion was reduced in the deletion strain and that ScCHD1 inhibited transcription. This observation, along with the known roles of other proteins with chromo or SNF2-related helicase/ATPase domains, suggests that alteration of gene expression by CHD genes might occur by modifications of chromatin structure, with altered access of the transcriptional apparatus to its chromosomal DNA template.

Figure 1

Amino acid alignments of selected regions of cloned human CHD proteins with MmCHD1. Identical residues are boxed in black and conserved residues shaded gray. Numbers represent residue positions within each sequence. Panels indicate regions of the alignment showing (a) chromo domains and selected segments of the (b) helicase/ATPase and (c) DNA-binding domains.

Figure 2

Northern blots for HsCHD1, HsCHD2, and HsCHD3. Lanes contain 2 μg poly(A)⁺ RNA extracted from heart (lane a), brain (lane b), placenta (lane c), lung (lane d), liver (lane e), skeletal muscle (lane f), kidney (lane g), and pancreas (lane h).

Figure 3

Protein domains found within CHD gene family members. Gene names are as described in the text. Extent of available sequence is indicated for GgCHD1-NW and DmCHD3; all other sequences are full length.

Figure 4

Clustering relationships between members of the CHD family. Multiple sequence alignment of region for which overlapping sequences were available were used with the phylip Phylogeny Inference Package to generate unrooted fitch trees. drawgram was used to plot the tree diagram. The scale bar indicates map units per unit branch length, as determined by fitch.

Figure 5

(A) Effect of 6AU on yeast colony growth. Values for colony growth are normalized as a percent of untreated values. Error bars show standard error of the mean. Circles represent wild-type cells (YMB669) and squares strains in which ScCHD1 was deleted (YMB592). Filled symbols represent strains that were transformed with ScCHD1 cloned into pRS425 (pRS425/CHD), a high-copy number vector, whereas open symbols denote transformation with empty vector pRS425. (B) Appearance of yeast colonies after growth on yeast extract/peptone/dextrose (YPD) plates (a and d) or plates containing 500 μg/ml (b and e) or 1,000 μg/ml 6AU (c and f). Plates were spread with either wild-type (a, b, and c) or ScCHD1 deletion strains (d, e, and f).