Human family with sequence similarity 60 member A (FAM60A) protein: a new subunit of the Sin3 deacetylase complex

Abstract

Here we describe the function of a previously uncharacterized protein, named family with sequence similarity 60 member A (FAM60A) that maps to chromosome 12p11 in humans. We use quantitative proteomics to determine that the main biochemical partners of FAM60A are subunits of the Sin3 deacetylase complex and show that FAM60A resides in active HDAC complexes. In addition, we conduct gene expression pathway analysis and find that FAM60A regulates expression of genes that encode components of the TGF-beta signaling pathway. Moreover, our studies reveal that loss of FAM60A or another component of the Sin3 complex, SDS3, leads to a change in cell morphology and an increase in cell migration. These studies reveal the function of a previously uncharacterized protein and implicate the Sin3 complex in suppressing cell migration.

Fig. 1.

FAM60A is a metazoan-specific Sin3/HDAC interacting protein. A, Heat map for the relative protein abundances expressed as dNSAF in the core Sin3/HDAC complex. The data are represented in a bait-prey matrix, where each column corresponds to purification of a bait protein and each row corresponds to a prey protein: values are average dNSAF for each protein. Color intensity represents protein abundance with brightest yellow indicating highest abundance and decreasing intensity indicating decreased abundance. Black indicates that the protein was not detected in the particular purification. Prey proteins were separated into two groups based on prior knowledge (i.e. known interactions colored in purple and new interactions colored in blue). B, Western blots showing FAM60A protein levels across several human cancer cell lines. MW = molecular weight marker. Beta-tubulin is used as a loading control; asterisks (*) denote potential modified forms of beta-tubulin. C, Silver stain analysis of FAM60A-FL complexes purified from 293T cells. D, Interaction network of the FAM60A protein and its associated proteins identified by MudPIT. The highest abundance proteins (top 22 sorted proteins) identified in two replicates of the FAM60A bait are illustrated in the figure. The bait protein is depicted as a hexagon, preys are denoted by squares. The color green denotes subunits of the Sin3/HDAC complex and the color brown indicates new associations with the FAM60A bait. Edges are colored based on the average dNSAF values. Black lines correspond to interactions with the highest abundance and red lines represent interactions with decreased abundance as shown in top left of (A). Identified proteins were grouped according to the functional classification. E, HDAC activity on ³H acetylated core histones with FAM60A-FL purified complexes or control purifications. 1 μm TSA was added to the deacetylation reaction where indicated.

Fig. 2.

Loss of FAM60A affects cell morphology and gene expression. A, A549 cells were transfected with control siRNAs, or siRNAs against FAM60A, SDS3, or ING2. B, Western blots of whole cell extracts from FAM60A, SDS3, and control knockdown using the indicated antibodies. C, The gene expression response of FAM60A knockdown relative to a nontargeting control is shown in the plot. The y-axis indicates the ratio of expression as log2(FAM60A knockdown/control), whereas the combined signal from both samples is plotted on the x-axis as 0.5 *(log2(FAM60A knockdown)+log2(control)). The top differentially expressed genes were selected by requiring an expression difference of at least 1.5-fold and applying an adjusted p value cut-off of 0.05. Probes corresponding to FAM60A are highlighted on the plot. D, A subset of go terms identified in microarray analysis in FAM60A knockdown cells.

Fig. 3.

FAM60A and SDS3 regulate common subsets of genes. A, Scatter plot showing the changes in gene expression in SDS3 knockdown cells relative to a nontargeting control siRNA. The y-axis indicates the ratio of expression as log2 (SDS3 knockdown/control), whereas the combined signal from both samples is plotted on the x-axis as 0.5 *(log2(SDS3 knockdown) + log2(control)). The top differentially expressed genes were selected by requiring an expression difference of at least 1.5-fold and applying an adjusted p value cut-off of 0.05. Probes corresponding to SDS3 are highlighted on the plot. B, The number of genes enriched in common on knockdown of either FAM60A or SDS3 is shown in the Venn diagram. The top gene set for each knockdown was identified using the criteria described in (A). The significance of the degree of overlap was assessed using the hypergeometric distribution and returned a p value of < 2.22e-16. C, Bar graph showing the genes that were up-regulated in both FAM60A and SDS3 knockdown cells with functions related to cell migration or extracellular matrix (ECM) structure. The fold-increase of the top probe for each gene is shown. A 1.5-fold cutoff was used and all genes shown have a p value of less than 1.67e-04.

Fig. 4.

Loss of FAM60A or SDS3 increases cell migration in A549 lung cancer cells. A, Migration assays in control, FAM60A, or SDS3 knockdown cells. A representative example is shown for each siRNA. B, Quantification of migration assays shown in (A). The average of at least triplicate biological replicates is shown. Error bars represent plus/minus one average deviation. C, Percent BrdU incorporation in A549 cells transfected with control siRNAs or siRNAs against FAM60A. Bars represent the average BrdU incorporation of three biological replicates, error bars represent ± one standard deviation.

Fig. 5.

FAM60A regulates cell morphology and migration of HepG2 cells. A, Morphology changes in HepG2 cells transfected with control siRNAs or siRNAs against FAM60A. B, Western blots of whole cell extracts from HepG2 knockdown cells using the indicated antibodies. C, Migration assays in HepG2 knockdown cells transfected with control siRNAs or siRNAs against FAM60A. A representative example is shown. D, Quantification of HepG2 migration assays. The average from several independent transfections is shown (control siRNA n = 5; FAM60A siRNA#1 n = 7; FAM60A siRNA#2 n = 4); error bars represent ± one standard deviation. The p value was calculated using a two-tailed t test. E, BrdU incorporation was measured in HepG2 cells transfected with control siRNAs or siRNAs against FAM60A. Bars represent the average of six independent transfections for each siRNA ± one standard deviation.

Fig. 6.

FAM60A directly represses expression of genes in the TGF-beta pathway. A, Genes increased by microarray analysis in FAM60A and SDS3 knockdown cells that encode components of the TGF-beta signaling pathway. Fold-increase of the top probe for that gene is shown. A 1.5-fold cutoff was used and all genes shown have a p value of less than 3.38e-05. B, C, Chromatin immunoprecipitation (ChIP) assays in control or FAM60A knockdown cells at the TGF-beta receptor 1 promoter (B) or SMAD2 promoter (C). Error bars represent ± one standard deviation of triplicate real-time PCR reactions. D, E, Migration assays in FAM60A knockdown cells in the presence of vehicle or SB-43152. A representative example for each siRNA against FAM60A is shown. F, Quantification of migration assays in FAM60A knockdown cells in the presence of vehicle (V) or SB-431542 (SB). Bars represent the average of at least three monitored scratches. Error bars represent plus/minus one average deviation. G, Western blots using the indicated antibodies in vehicle treated FAM60A knockdown cells and SB-431542 treated knockdown cells.