C/EBPδ gene targets in human keratinocytes

Abstract

C/EBPs are a family of B-Zip transcription factors--TFs--involved in the regulation of differentiation in several tissues. The two most studied members--C/EBPα and C/EBPβ--play important roles in skin homeostasis and their ablation reveals cells with stem cells signatures. Much less is known about C/EBPδ which is highly expressed in the granular layer of interfollicular epidermis and is a direct target of p63, the master regular of multilayered epithelia. We identified C/EBPδ target genes in human primary keratinocytes by ChIP on chip and profiling of cells functionally inactivated with siRNA. Categorization suggests a role in differentiation and control of cell-cycle, particularly of G2/M genes. Among positively controlled targets are numerous genes involved in barrier function. Functional inactivation of C/EBPδ as well as overexpressions of two TF targets--MafB and SOX2--affect expression of markers of keratinocyte differentiation. We performed IHC on skin tumor tissue arrays: expression of C/EBPδ is lost in Basal Cell Carcinomas, but a majority of Squamous Cell Carcinomas showed elevated levels of the protein. Our data indicate that C/EBPδ plays a role in late stages of keratinocyte differentiation.

Figure 1. Identification of C/EBPδ targets by ChIP on chip.

A. Generation of the ChIP amplicons for ChIP on chip experiments. ChIP of human primary keratinocytes with C/EBPδ and control antibodies. The known targets tested are indicated. The normalization of the immunoprecipitated material was performed by evaluating a negative genomic region (Centromeric Satellite 11). B. Gene Ontology analysis of C/EBPδ targets genes from the ChIP on chip screening.

Figure 2. Validation of C/EBPδ targets in primary keratinocytes.

A. Analysis of the loci targeted derived from the ChIP on chip experiments by qPCR analysis of ChIPs with anti-C/EBPδ and control antibodies from human primary keratinocytes. B. The position of the ampolicons (black bars) and of the putative C/EBP consensus site (circle) are indicated for targets analyzed in A.

Figure 3. Identification of C/EBPδ-regulated genes in human keratinocytes.

A. Left Panels, RT-PCR analysis of human primary keratinocytes after C/EBPδ RNAi inactivation at 48 hours post-transfection. cDNA normalization was performed with GAPDH. Right Panels, Western Blot analysis of the same C/EBPδ-inactivated human primary keratinocytes with the C/EBPδ antibody. Vinculin was used as a loading control. B. Heat map showing the mRNA expression levels of several classes of genes after C/EBPδ inactivation. C. qRT-PCR analysis of C/EBPδ regulated genes that emerged from the expression profiling. D. ChIP analysis of promoter regions of C/EBPδ-regulated genes with chromatin from human primary keratinocytes, with α-C/EBPδ and control antibodies. In the Left Panel, adult and neonatal keratinocytes were analyzed in semi-quantitative PCR. In the Right Panel, the targets were validated by qPCR in adult keratinocytes.

Figure 4. C/EBPδ and p63 regulated genes.

Genes coregulated by p63 and C/EBPδ, either activated or repressed (Table S2). The data on p63 were derived from References 32, 36–39.

Figure 5. C/EBPδ is important for keratinocyte differentiation.

Analysis of differentiation markers by qRT-PCR of C/EBPδ-inactivated cells induced to differentiate. Primary keratinocytes were transfected with scamble and C/EBPδ siRNAs. After 24 h, half of the cells were induced to differentiate by addition of calcium. Samples were harvested after 3 days, RNAs prepared and qPCR performed on the indicated genes. Values are normalized to GAPDH, used as an internal control.

Figure 6. Role of C/EBPδ target genes in keratinocyte differentiation.

A. Analysis of differentiation markers by qRT-PCR of primary keratinocytes induced to differentiate after transient transfection of MafB expression plasmid and empty vector as control. After 24 h, half of the cells were induced to differentiate by addition of calcium, as in Fig. 5. B. Same as A, except that Sox2 was overexpressed.

Figure 7. Overexpression of C/EBPδ in human non-melanoma skin tumors.

Immunohistochemistry analysis of p63 and C/EBPδ in normal skin (Upper Panel), BCC (Middle Panel) and SCC (Lower Panel).

Figure 8. Scheme of C/EBPδ targets in human skin.