Skin is the largest organ in the body and serves important barrier, regulatory, and sensory functions. The epidermal layer shows rhythmic physiological responses to daily environmental variation (e.g., DNA repair). We investigated the role of the circadian clock in the transcriptional regulation of epidermis using a hybrid experimental design, in which a limited set of human subjects (n = 20) were sampled throughout the 24-h cycle and a larger population (n = 219) were sampled once. We found a robust circadian oscillator in human epidermis at the population level using pairwise correlations of clock and clock-associated genes in 298 epidermis samples. We then used CYCLOPS to reconstruct the temporal order of all samples, and identified hundreds of rhythmically expressed genes at the population level in human epidermis. We compared these results with published time-series skin data from mice and found a strong concordance in circadian phase across species for both transcripts and pathways. Furthermore, like blood, epidermis is readily accessible and a potential source of biomarkers. Using ZeitZeiger, we identified a biomarker set for human epidermis that is capable of reporting circadian phase to within 3 hours from a single sample. In summary, we show rhythms in human epidermis that persist at the population scale and describe a path to develop robust single-sample circadian biomarkers.

Preterm birth (PTB) complications are the leading cause of long-term morbidity and mortality in children. By using whole blood samples, we integrated whole-genome sequencing (WGS), RNA sequencing (RNA-seq), and DNA methylation data for 270 PTB and 521 control families. We analyzed this combined dataset to identify genomic variants associated with PTB and secondary analyses to identify variants associated with very early PTB (VEPTB) as well as other subcategories of disease that may contribute to PTB. We identified differentially expressed genes (DEGs) and methylated genomic loci and performed expression and methylation quantitative trait loci analyses to link genomic variants to these expression and methylation changes. We performed enrichment tests to identify overlaps between new and known PTB candidate gene systems. We identified 160 significant genomic variants associated with PTB-related phenotypes. The most significant variants, DEGs, and differentially methylated loci were associated with VEPTB. Integration of all data types identified a set of 72 candidate biomarker genes for VEPTB, encompassing genes and those previously associated with PTB. Notably, PTB-associated genes RAB31 and RBPJ were identified by all three data types (WGS, RNA-seq, and methylation). Pathways associated with VEPTB include EGFR and prolactin signaling pathways, inflammation- and immunity-related pathways, chemokine signaling, IFN-γ signaling, and Notch1 signaling. Progress in identifying molecular components of a complex disease is aided by integrated analyses of multiple molecular data types and clinical data. With these data, and by stratifying PTB by subphenotype, we have identified associations between VEPTB and the underlying biology.

One Sentence Summary: Human epidermis shows strong circadian rhythms at the population scale and provides a better source for developing robust, single-sample circadian phase biomarkers than human blood. Abstract:Skin is the largest organ in the body and serves important barrier, regulatory, and sensory functions. Like other tissues, skin is subject to temporal fluctuations in physiological responses under both homeostatic and stressed states. To gain insight into these fluctuations, we investigated the role of the circadian clock in the transcriptional regulation of epidermis using a hybrid experimental design, where a limited set of human subjects (n=20) were sampled throughout the 24 h cycle and a larger population (n=219) were sampled once. By looking at pairwise correlations of core clock genes in 298 skin samples, we found a robust circadian oscillator in skin at the population level. Encouraged by this, we used CYCLOPS to reconstruct the temporal order of all samples and identified hundreds of rhythmically-expressed genes at the population level in human skin. We compared these results with published time-series skin data from mouse and show strong concordance in circadian phase across species for both transcripts and pathways. Further, like blood, skin is readily accessible and a potential source of biomarkers.Using ZeitZeiger, we identified a biomarker set for human skin that is capable of reporting circadian phase to within 3 h from a single sample. In summary, we show rhythms in human skin that persist at the population scale and a path to develop robust single-sample circadian biomarkers.

Retinoic acid is an embryonic morphogen and dietary factor that demonstrates chemotherapeutic efficacy in inducing maturation in leukemia cells. Using HL60 model human myeloid leukemia cells, where all-trans retinoic acid (RA) induces granulocytic differentiation, we developed two emergent RA-resistant HL60 cell lines which are characterized by loss of RA-inducible G1/G0 arrest, CD11b expression, inducible oxidative metabolism and p47phox expression. However, RA-treated RA-resistant HL60 continue to exhibit sustained MEK/ERK activation, and one of the two sequentially emergent resistant lines retains RA-inducible CD38 expression. Other signaling events that define the wild-type (WT) response are compromised, including c-Raf phosphorylation and increased expression of c-Cbl, Vav1, and the Src-family kinases (SFKs) Lyn and Fgr. As shown previously in WT HL60 cells, we found that the SFK inhibitor PP2 significantly increases G1/G0 cell cycle arrest, CD38 and CD11b expression, c-Raf phosphorylation and expression of the aforementioned regulators in RA-resistant HL60. The resistant cells were potentially incapable of developing inducible oxidative metabolism. These results motivate the concept that RA resistance can occur in steps, wherein growth arrest and other differentiation events may be recovered in both emergent lines. Investigating the mechanistic anomalies in resistant cell lines is of therapeutic significance and helps to mechanistically understand the response to retinoic acid’s biological effects in WT HL60 cells.

In this study, we present an effective model All-Trans Retinoic Acid (ATRA)-induced differentiation of HL-60 cells. The model describes reinforcing feedback between an ATRA-inducible signalsome complex involving many proteins including Vav1, a guanine nucleotide exchange factor, and the activation of the mitogen activated protein kinase (MAPK) cascade. We decomposed the effective model into three modules; a signal initiation module that sensed and transformed an ATRA signal into program activation signals; a signal integration module that controlled the expression of upstream transcription factors; and a phenotype module which encoded the expression of functional differentiation markers from the ATRA-inducible transcription factors. We identified an ensemble of effective model parameters using measurements taken from ATRA-induced HL-60 cells. Using these parameters, model analysis predicted that MAPK activation was bistable as a function of ATRA exposure. Conformational experiments supported ATRA-induced bistability. Additionally, the model captured intermediate and phenotypic gene expression data. Knockout analysis suggested Gfi-1 and PPARg were critical to the ATRAinduced differentiation program. These findings, combined with other literature evidence, suggested that reinforcing feedback is central to hyperactive signaling in a diversity of cell fate programs.