Conservation of transcriptional regulation by BRCA1 and BARD1 in Caenorhabditis elegans

Abstract

The tumor-suppressor proteins BRCA1 and BARD1 function as an E3 ubiquitin ligase to facilitate transcriptional repression and DNA damage repair. This is mediated in-part through its ability to mono-ubiquitylate histone H2A in nucleosomes. Studies in Caenorhabditis elegans have been used to elucidate numerous functions of BRCA1 and BARD1; however, it has not been established that the C. elegans orthologs, BRC-1 and BRD-1, retain all the functions of their human counterparts. Here we explore the conservation of enzymatic activity toward nucleosomes which leads to repression of estrogen-metabolizing cytochrome P450 (cyp) genes in humans. Biochemical assays establish that BRC-1 and BRD-1 contribute to ubiquitylation of histone H2A in the nucleosome. Mutational analysis shows that while BRC-1 likely binds the nucleosome using a conserved interface, BRD-1 and BARD1 have evolved different modes of binding, resulting in a difference in the placement of ubiquitin on H2A. Gene expression analysis reveals that in spite of this difference, BRC-1 and BRD-1 also contribute to cyp gene repression in C. elegans. Establishing conservation of these functions in C. elegans allows for use of this powerful model organism to address remaining questions regarding regulation of gene expression by BRCA1 and BARD1.

Figure 1.

H2A ubiquitylation is a conserved feature of CeBCBD, but does not primarily occur at the predicted location. (A) Amino acid sequence alignment of worm (Ce) and human (Hs) histone H2A tails. The residue highlighted in black, K125, is the lysine closest to the three residues targeted by HsBCBD shown in bold. Dashes indicate gaps in the sequence. (B) In vitro activity of CeBCBD and HsBCBD toward nucleosomes containing H2A with the worm C-terminal tail sequence (WT) or K125H mutation is demonstrated via western blot for H2A. (C) Mean unmodified H2A remaining after 10 and 30 min from two biological and two technical replicates of in vitro H2A ubiquitination assays. Error bars represent the standard deviation. The intensity of unmodified H2A bands in western blots such as (B) were quantified using ImageJ. Means were compared using a Welch's t-test for data with unequal variance (HsBCBD at 30 min) or an unpaired two-sample t-test was used for data with equal variances (all other time points). Statistical significance was determined using a P-value <0.05. An uncropped blot image is available in the supplementary information.

Figure 2.

BRC-1 utilizes a conserved nucleosome binding interface. (A) Sequence alignments of the relevant regions of the RING domains and solution structure (PDB 1JM7) of HsBCBD highlighting the sidechains of residues from BRCA1 and BARD1 that are important for binding to the nucleosome in orange and red, respectively. The sidechain of BARD1 R99 that non-covalently engages ubiquitin in E2∼Ub is shown in blue. Zinc coordinating residues that were mutated in families with a history of breast cancer are shown in gray. Western blots comparing the ubiquitylation activity of mutant BRC-1 (B) and mutant BRD-1 (D) constructs to wild-type CeBCBD (WT). The substrate observed is H2A incorporated in nucleosomes. Means and standard deviations of unmodified H2A at 30 min are presented for BRC-1 mutants (C) and BRD-1 mutants (E). Asterisk denotes mutants with significant decreases in activity according to Student's t-test with P-value < 0.05, while ‘ns’ denotes activity difference is not significant (P-value > 0.05). (F) BRD-1 homology model generated by SWISS-MODEL (light blue) highlighting the K54 and R55 basic patch in green (44). The additional loop in BRD-1 that contains these residues is hypothesized to be positioned below the nucleosome binding residues for BARD1 (red). Uncropped blot images are available in the supplementary information.

Figure 3.

CeBCBD regulates the expression of cyp-13A genes in vivo. The expression of cyp-13A2, cyp-13A4, cyp-13A5, cyp-13A6, cyp-13A7, cyp-13A8, cyp-13A10, cyp-13A11 and cyp-13A12 in WT (N2) and experimental worm strains Δbrc-1 (gk5332) (A) and Δbrd-1 (dw1) (B) were measured with RT-qPCR. All the data are presented as the mean starting quantity (SQ) normalized to reference gene tba-1 and presented relative to simultaneously measured WT SQ. Each error bar represents ± SEM and * denotes statistically significant deviation from WT as determined by Student's t-test. P-values <0.05 were considered statistically significant.