Network Analysis Identifies Mitochondrial Regulation of Epidermal Differentiation by MPZL3 and FDXR

Abstract

Current gene expression network approaches commonly focus on transcription factors (TFs), biasing network-based discovery efforts away from potentially important non-TF proteins. We developed proximity analysis, a network reconstruction method that uses topological constraints of scale-free, small-world biological networks to reconstruct relationships in eukaryotic systems, independent of subcellular localization. Proximity analysis identified MPZL3 as a highly connected hub that is strongly induced during epidermal differentiation. MPZL3 was essential for normal differentiation, acting downstream of p63, ZNF750, KLF4, and RCOR1, each of which bound near the MPZL3 gene and controlled its expression. MPZL3 protein localized to mitochondria, where it interacted with FDXR, which was itself also found to be essential for differentiation. Together, MPZL3 and FDXR increased reactive oxygen species (ROS) to drive epidermal differentiation. ROS-induced differentiation is dependent upon promotion of FDXR enzymatic activity by MPZL3. ROS induction by the MPZL3 and FDXR mitochondrial proteins is therefore essential for epidermal differentiation.

Keywords: FDXR; MPZL3; differentiation; mitochondria; network biology.

Figure 1. Proximity Analysis Identifies MPZL3 as a Highly Connected Hub in an Epidermal Differentiation Network

(A) Proximity Analysis workflow consists of a correlation matrix generation that uses topological constraints of scale-free small-world networks to identify genes that are highly connected, and subsequently cutaneous SCC data is used to highlight potential regulators of differentiation. (B) Network depiction of top 10,000 edges from the network. (C) Gene ontology terms for the 500 most connected genes in the differentiation network. (D) Top 6 candidates that emerge from proximity analysis and their characteristics in terms of dynamic hub score, known function and disease relevance. (E) Expression of MPZL3 in differentiation datasets (n = 14) when compared to subconfluent keratinocytes and in cutaneous SCC (n=21) when compared to matched normal skin, mean +/− SD shown. (F) mRNA levels by qPCR from laser capture microscopy separation of basal and suprabasal layers of the skin. (G) Immunofluorescent staining of MPZL3 (red) and collagen VII (green) in normal human skin [left] as well as a no MPZL3 antibody control stained with collagen VII [right]. Bar 15 μM. See also Figures S1 and S2 and Tables S1, S2 and S3.

Figure 2. MPZL3 is Required for Normal Epidermal Differentiation

(A) Knockdown of MPZL3 with two distinct siRNAs in an organotypic model of differentiation indicates that markers of differentiation (K1, K10, LOR, TGM1) are not expressed as highly in MPZL3i samples compared to a control siRNA. Bar 20 μM. (B) Quantification of transcript levels of a panel of epidermal differentiation markers with either control siRNA or MPZL3 siRNA mediated KD. n = 3 biological replicates, mean +/− SEM is shown. (C) Rescue experiments of the MPZL3 KD phenotype was evaluated in the context of forced expression of wild type MPZL3, MPZL3 R99Q, or two truncated versions of MPZL3 with siRNAs to KD MPZL3. Values shown in heatmap are the mean of 3 biological replications, averaged between 2 siRNAs. (D) RNA-Sequencing was performed on keratinocytes that were subjected to differentiation conditions in culture. Heatmap shows genes that are changed at least 4 fold in either of the MPZL3 KD conditions compared to control siRNA. (E) Gene ontology analysis of the genes that are down regulated in the RNA-sequencing dataset with MPZL3 KD. (F) Overlap of the genes that are down regulated with MPZL3 KD with the gene signatures of known regulators of epidermal differentiation. The left column indicates the number of genes involved in the overlap and [***] indicates a p-value < 0.001 (Fisher's exact test), [ns] indicates a non-significant p-value. (G) MPZL3 expression level in datasets with perturbation of known regulators of epidermal differentiation (Boxer et al., 2014; Hopkin et al., 2012; Kretz et al., 2013; Lopez-Pajares et al., 2015; Truong et al., 2006). (H) ChIP qPCR analysis of genomic regions surrounding MPZL3 genomic locus indicates known regulators of MPZL3 transcript expression are enriched for binding in a proximal genomic region compared to an IgG control. n = 2 biological replicates, mean +/− SD shown. (I) Quantification of transcript levels in heatmap format of a panel of epidermal differentiation markers with the KD of known regulators of epidermal differentiation, with or without forced expression of MPZL3. See also Figure S2 and Table S4.

Figure 3. MPZL3 Localizes in Mitochondria and Interacts with FDXR

(A) Depiction of MPZL3 proximal proteins identified by mass spectrometry of biotinylated proteins isolated from differentiated cells expressing an MPZL3-BirA* fusion construct. The 53 proteins shown in this network were detected exclusively in the MPZL3-BirA* expressing cells as opposed to BirA* only expressing cells and were filtered for proteins that are commonly found in BirA* experiments. (B) Electron microscopy in keratinocytes expressing an MPZL3 overexpression construct. Antibodies to HA were used to identify MPZL3 and 5 nm gold conjugate was used for detection. Representative image of a mitochondria dotted with gold particles shown. (C) Quantification of observed localization of gold conjugates tagging the expressed MPZL3-FHH protein. (D) Table representing the top proximal hits from the MPZL3-BirA* tagging experiment indicating number of spectral counts for each hit as well as the SAINT score. (E) Proximity ligation analysis with endogenous MPZL3 and FDXR. Signal representing the interaction is shown in red and nuclei are marked by blue DAPI signal. siRNAs targeting either MPZL3 or FDXR ablate the observed signal. Representative images are shown. Bar 7 μM. (F) Co-immunoprecipitation between MPZL3 and FDXR; 1% of input is shown. (G) Proximity ligation analysis on HA-tagged constructs of wild type MPZL3, MPZL3 R99Q, and two truncations of MPZL3 with endogenous FDXR antibody. Signal representing the interaction is shown in red and nuclei are marked by blue DAPI signal. Representative images are shown. Bar 7 μM. (H) Quantification of the proximity ligation analysis signal shown in (G). n=3 biological replicates with at least 10 images analyzed per replicate, mean +/− SEM is shown. See also Figure S3 and S4 and Table S5.

Figure 4. FDXR phenocopies and rescues MPZL3 differentiation defects

(A) Knockdown of FDXR with two distinct siRNAs in human organotypic epidermis indicates that markers of differentiation (K1, K10, LOR, TGM1) are inhibited in FDXR KD samples compared to control siRNA. Bar 20 μM. (B) Quantification of transcript levels of a panel of epidermal differentiation markers with either control or FDXR siRNA mediated KD. n = 3 biological replicates, mean +/− SEM is shown. (C) RNA-Sequencing was performed on keratinocytes subjected to differentiation conditions in culture. Heatmap shows genes that are changed at least 4 fold in either of the FDXR KD conditions compared to control siRNA. (D) Overlap between genes changed with MPZL3 KD or FDXR KD. The overlap of 113 genes is significant with a p-value < 0.05, Fisher's exact test. (E) Gene ontology analysis of the set of overlapped genes. (F) Quantitation of transcript levels of a panel of differentiation markers in subconfluent cells with enforced expression of FDXR. (G) Rescue experiments of the MPZL3 KD phenotype in the context of forced expression of FDXR with siRNAs to KD MPZL3. Quantitation of transcript levels of a panel of epidermal differentiation markers is shown, n = 3 biological replicates, mean +/− SEM is shown. See also Table S6.

Figure 5. MPZL3 and FDXR Regulate and are Required for ROS Mediated Differentiation

(A) Reactive oxygen species (ROS) levels in differentiating cultured keratinocytes as measured by DCF-DA fluorescence. Hydrogen peroxide (H202) was used as a measurement control. [*] indicates p <0.05. n = 5 biological replicates, mean +/− SEM is shown. (B) ROS levels with MPZL3 and/or FDXR KD as measured by DCF-DA fluorescence. [*] indicates p <0.05. n = 5 biological replicates, mean +/− SEM is shown. (C) ROS levels with forced MPZL3 and/or FDXR expression as measured by DCF-DA fluorescence. [*] indicates p <0.05. n = 5 biological replicates, mean +/− SEM is shown. (D) Quantitation of transcript levels of a panel of differentiation markers with KD of MPZL3 and FDXR by siRNA with either DMSO treatment or treatment with galactose oxidase (GAO). n = 3 biological replicates, mean +/− SD is shown. [*] indicates p <0.05, [**] p < 0.01, [***] p < 0.001. (E) Quantitation of transcript levels of a panel of differentiation markers with enforced expression of MPZL3 and FDXR with either DMSO treatment or treatment with EUK134. n = 3 biological replicates, mean +/− SD is shown. (H) Summary heatmap of the ROS rescue experiments. See also Supplemental Figure S5.

Figure 6. FDXR Enzymatic Activity is Required for its Role in Differentiation

(A) Relative amount of NADPH as measured by fluorescence compared to differentiated keratinocytes treated with empty vector (EV). n = 3 biological replicates, mean +/− SEM is shown (B) Relative amount of NADPH as measured by fluorescence compared to differentiated keratinocytes treated with EV. Mutant FDXR constructs marked in red are predicted to perturb NADPH binding by FDXR and those in blue are predicted to perturb FAD binding by FDXR. n = 3 biological replicates, mean +/− SEM is shown (C) Quantitation of transcript levels of a panel of differentiation markers in subconfluent keratinocytes subjected to forced expression of wild-type or mutant FDXR constructs relative to EV. n = 3 biological replicates, mean +/− SEM is shown (D) Rescue experiments of the MPZL3 KD phenotype were evaluated in the context of forced expression of FDXR or FDXR mutants with siRNAs to KD MPZL3. Quantitation of transcript levels of a panel of epidermal differentiation markers is shown in heatmap format, n = 3 biological replicates, mean +/− SD is shown. (E) Model of transcriptional regulation of MPZL3 by known regulators of epidermal differentiation, followed by mitochondrial localization, interaction with FDXR and joint regulation of ROS required for terminal epidermal differentiation. See also Figure S6.