Regulator of sex-limitation KRAB zinc finger proteins modulate sex-dependent and -independent liver metabolism

Abstract

Krüppel-related zinc finger proteins (KRAB-zfps) comprise the largest mammalian transcription factor family, but their specific functions are largely unknown. Two KRAB-zfps, regulator of sex-limitation (Rsl) 1 and Rsl2, repress expression of the mouse sex-limited protein (Slp) gene, the hallmark of Rsl activity, as well as some other male-predominant liver genes. This phenotype suggests Rsl modifies sex-specific transcription. The scope of Rsl control was determined by expression profiling of liver RNA from wild-type (wt), rsl, and transgenic mice with hepatic overexpression of Rsl1 or Rsl2. About 7.5% of the liver transcriptome was Rsl-responsive. More genes in males than females were affected by the loss of Rsl (e.g., in rsl mice), whereas Rsl overexpression altered more transcripts in females than males. Rsl dramatically repressed some female-predominant genes, but most were modestly (1.25- to 2-fold) influenced. In males, most Rsl-responsive genes unexpectedly expressed at lower levels in rsl than wt, suggesting not all are direct targets of Rsl repression. Gene Ontology analysis showed Rsl targets enriched in pathways of cholesterol, steroid, and lipid metabolism, linking Rsl to energy balance. In accord with this, blood glucose levels were less in male rsl than wt mice, and less responsive to fasting and refeeding. rsl mice were also leaner than wt, consistent with their hepatic regulation of phosphoenolpyruvate carboxykinase 1 and stearoyl-Coenzyme A desaturase 1. Altogether, Rsl's effect on sexually dimorphic and metabolically sensitive liver gene expression suggests a role for KRAB-zfps as broad genetic modulators of individual adaptation.

Fig. 1.

Supraphysiological levels of liver-specific regulator of sex limitation (Rsl) extinguish sex-limited protein (Slp) expression in both sexes. A: semiquantitative RT-PCR indicates tg expression is ∼100-fold higher than the endogenous genes. Unlike the homozygous wild-type (wt) and rsl mice, the transgenic (tg) mice analyzed here (R1 and R2) are heterozygous at the Rsl locus. Gross overexpression from the transgenes in these mice had little influence on the endogenous levels of Rsl1 or Rsl2. B: Slp expression is dramatically repressed in mice with excess Rsl. Slp mRNA was assayed by real-time quantitative (Q)-RT-PCR and values were normalized to wt males. Slp was virtually undetectable in RNA from Rsl females, Rsl1 tg females, and both sexes of Rsl2 tg mice. Bars represent means ± SE. Rsl males, n = 5; Rsl females, n = 6; rsl males, n = 5; rsl females, n = 7; each sex of tg mice, n = 6.

Fig. 2.

Expression profiling of RNA from wt, rsl, and transgenic mouse liver. A: false-color heat map highlighting sex-specific gene expression in wt and rsl liver. We found 20,393 probe sets surpassed the minimum expression threshold and were arranged according to their magnitude of sex-biased expression determined by the male-to-female ratio of their average expression values. Male-predominant genes are in green, female-predominate genes are in red, and the magnitude of the expression ratio is according to the color bar shown at bottom. At the extremes are the Y-chromosome Ddx3y and X-chromosome Xist genes. Twenty-four probe sets appear inverted in their sex specificity in rsl relative to wt mice (*). The greater percentage of black in the rsl column (column 2) indicates that liver gene expression is less sex-biased in rsl than in wt mice. The same arrangements of probe sets were similarly color coded for wt/rsl male and wt/rsl female comparisons. B: false color heat maps of probe sets that differ from wt mice in at least 1 mouse genotype (P < 0.05). To visualize sex-dependent expression patterns, the probe sets were organized independently in males and females. The color intensity reflects the abundance of transcript relative to the spot average. Red is below the mean, and green is above the mean, with fold deviation in accord with the color bar at bottom. Labels below the columns identify results from duplicate pools [A (left) and B (right)]. The dendrograms at top, also highlighted by the vertical white lines, indicate that tg mice (R1 and R2) retained an overall pattern of expression similar to rsl rather than wt males, whereas in females, tg mice were similar to each other but differed considerably from either nontransgenic parental strain. C: Venn diagrams showing the distribution of expression patterns in B across genotypes. Males are on the left; females are on the right.

Fig. 3.

Real-time Q-RT-PCR validation of sexually dimorphic Rsl-responsive genes identified by microarray. Female-predominant genes are on top and male-predominant genes are below. Above the graphs are close-up images taken from the heat maps in Fig. 2B. To simplify the comparison, the color intensity of a single bar per genotype was adjusted to approximate the mean of pool A and pool B. Below the heat maps are real-time Q-RT-PCR results normalized to the mean expression in wt mice. Sex-specific expression ratios in wt mice (top, female/male; bottom, male/female) are in the top left corner. Bars represent means ± SE. *Mice that differ from rsl, *P < 0.05 and **P < 0.01. wt mice, n = 10; rsl males, n = 10; rsl females, n = 9; tg males, n = 10, Rsl1-tg females, n = 17, Rsl2-tg females, n = 13.

Fig. 4.

Scatter plots to determine correlation of sex-biased liver gene expression with the presence or absence of Rsl. A: the log₂ ratios for the 907 Rsl-responsive probe sets in males were plotted according to their sex-biased expression in wt mice (x-axis) relative to their differential expression in wt vs. rsl mice (y-axis). Only genes for which the wt to rsl ratio is >1.5-fold are shown. Female-predominant genes are on the left (quadrants I and III). Male-predominant genes are on the right (quadrants II and IV). The shaded region highlights genes that are sex-neutral (< 1.5-fold Δ males v. females). Slp was not on the microarray but is plotted here for reference with relative expression values determined by real-time Q-RT-PCR. The trend line indicates the best-fit linear representation of the 395 probe sets. The low correlation coefficient (r = 0.52) suggests male-specific gene expression is not tightly linked to the presence of Rsl. The majority of points (67%) in quadrant IV suggest that most male-predominant liver genes in males, unlike Slp, are diminished in expression due to the loss of Rsl. B: the log₂ ratios for the 811 Rsl-responsive probe sets in females were plotted according to their sex-biased expression in wt and rsl mice. Axes, shading, and labeling are as in A. The majority of points (44%) in quadrant II suggest that nearly half of all male-predominant liver genes in females, similar to Slp, are increased in expression due to the loss of Rsl.

Fig. 5.

Comparison of genes responsive to Rsl and/or STAT5. The 492 Rsl-responsive transcripts were compared by GenBank accession number to the STAT5-responsive transcripts reported by Clodfelter et al. 2006 (7) and Clodfelter et al. 2007 (8). The overlapping gene numbers may be slightly underestimated due to differences in accession numbers for identical genes on the Affymetrix array, used here, and the Agilent Rosetta/Merck Mouse TOE 75k Array I used elsewhere (7, 8). For all data sets, significant difference thresholds were 1.5-fold (P < 0.05). While each study was comprehensive, the total number of transcripts analyzed differed slightly. Here we observed 20,393 unique liver transcripts. The STAT5a study reported 18,204, and the STAT5b study reported 23,455.

Fig. 6.

Metabolic homeostasis differs between wt and rsl mice. Body weight (n = 9 for each sex and genotype, A) and percent body fat (n = 4 for each sex and genotype, B) were measured in age-matched adults of both sexes of wt and rsl mice. C: blood glucose levels were measured at 7:30 PM (late in the “lights-on” phase) in wt and rsl mice, fed ad libitum (n = 9). Cohorts of wt and rsl mice (n = 3) were fasted for 12 h or given chow for 4 h after fasting. D: expression of Pck1 and Scd1, in mice of each sex and genotype, fed ad libitum (n = 7), fasted (n = 3), or fasted and refed (n = 3). Real-time Q-RT-PCR measured mRNA levels and values were normalized to wt males fed ad libitum. All bars represent means ± SE. †P < 0.10, *P < 0.05, wt vs. rsl. E: validation of changes in Scd1 protein. Representative females with relative Scd1 mRNA values near the mean were assayed by Western blot for Scd1 protein in liver extracts. For relative quantification, Scd1 band intensities were normalized to GAPDH.

Fig. 7.

Rsl-dependent liver gene regulation. Examples of targets that are likely regulated by direct repression include Slp, Sult3a1, Fmo3, and Cyp3a41. Genes such as Ugt2b38, Thrsp, and Angptl4 had expression patterns similar to Slp (i.e., higher in rsl than wt mice) but were only partially restored to wt levels by Rsl overexpression. Unexpected was the large proportion of genes that had the inverse expression pattern (i.e., lower in rsl than wt mice and elevated by Rsl overexpression) (e.g., Elovl3, Asns, Hsd3b5, etc.). Together these latter groups are likely controlled by indirect regulation via activators or repressors in pathways downstream from Rsl repression.