We characterized the mutational landscape of melanoma, the form of skin cancer with the highest mortality rate, by sequencing the exomes of 147 melanomas. Sun-exposed melanomas had markedly more ultraviolet (UV)-like C>T somatic mutations compared to sun-shielded acral, mucosal and uveal melanomas. Among the newly identified cancer genes was PPP6C, encoding a serine/threonine phosphatase, which harbored mutations that clustered in the active site in 12% of sun-exposed melanomas, exclusively in tumors with mutations in BRAF or NRAS. Notably, we identified a recurrent UV-signature, an activating mutation in RAC1 in 9.2% of sun-exposed melanomas. This activating mutation, the third most frequent in our cohort of sun-exposed melanoma after those of BRAF and NRAS, changes Pro29 to serine (RAC1P29S) in the highly conserved switch I domain. Crystal structures, and biochemical and functional studies of RAC1P29S showed that the alteration releases the conformational restraint conferred by the conserved proline, causes an increased binding of the protein to downstream effectors, and promotes melanocyte proliferation and migration. These findings raise the possibility that pharmacological inhibition of downstream effectors of RAC1 signaling could be of therapeutic benefit.

We report on whole-exome sequencing (WES) of 213 melanomas. Our analysis established NF1, encoding a negative regulator of RAS, as the third most frequently mutated gene in melanoma, after BRAF and NRAS. Inactivating NF1 mutations were present in 46% of melanomas expressing wild-type BRAF and RAS, occurred in older patients and showed a distinct pattern of co-mutation with other RASopathy genes, particularly RASA2. Functional studies showed that NF1 suppression led to increased RAS activation in most, but not all, melanoma cases. In addition, loss of NF1 did not predict sensitivity to MEK or ERK inhibitors. The rebound pathway, as seen by the induction of phosphorylated MEK, occurred in cells both sensitive and resistant to the studied drugs. We conclude that NF1 is a key tumor suppressor lost in melanomas, and that concurrent RASopathy gene mutations may enhance its role in melanomagenesis.

During mitosis, RNA polymerase II (Pol II) and many transcription factors dissociate from chromatin, and transcription ceases globally. Transcription is known to restart in bulk by telophase, but whether de novo transcription at the mitosis-G1 transition is in any way distinct from later in interphase remains unknown. We tracked Pol II occupancy genome-wide in mammalian cells progressing from mitosis through late G1. Unexpectedly, during the earliest rounds of transcription at the mitosis-G1 transition, ∼50% of active genes and distal enhancers exhibit a spike in transcription, exceeding levels observed later in G1 phase. Enhancer-promoter chromatin contacts are depleted during mitosis and restored rapidly upon G1 entry but do not spike. Of the chromatin-associated features examined, histone H3 Lys27 acetylation levels at individual loci in mitosis best predict the mitosis-G1 transcriptional spike. Single-molecule RNA imaging supports that the mitosis-G1 transcriptional spike can constitute the maximum transcriptional activity per DNA copy throughout the cell division cycle. The transcriptional spike occurs heterogeneously and propagates to cell-to-cell differences in mature mRNA expression. Our results raise the possibility that passage through the mitosis-G1 transition might predispose cells to diverge in gene expression states.

Rh alloimmunization remains a challenge for patients with sickle cell disease (SCD) despite transfusion of serologic Rh C, E, and K antigen-matched red cells. Inheritance of altered alleles contributes to the prevalence of Rh antibodies after blood transfusion in patients with SCD and explains approximately one-third of cases. The remainder seem to be stimulated by altered Rh proteins on African American donor red cells. Matching patients with donors on the basis of genotype may mitigate Rh alloimmunization, but the feasibility and resources required are not known. We compared allele frequencies between patients with SCD (n = 857) and African American donors (n = 587) and showed that allele frequencies are similar. Overall, 29% of and 53% of alleles are altered in patients and African American donors. We modeled genotype matching compared with serologic Rh D, C, and E, along with K antigen matching, and found that approximately twice the number of African American donors would be required for genotype vs Rh serologic matching at our institution. We demonstrated that African American donor recruitment is necessary to maintain an adequate supply of C-, E-, and K-negative donor units to avoid depleting the Rh-negative (RhD) blood supply. Our results suggest that prophylactic genetic matching for patients with SCD is feasible with a donor pool comprised primarily of African-Americans and would optimize the use of our existing minority donor inventory. The current cost of genotyping all minority donors and management of the data remain limiting factors.

Summary S-acylation, S-glutathionylation, S-nitrosylation, and S-sulfenylation are prominent, chemically distinct, modifications that regulate protein function, redox-sensing, and trafficking. Although the biological significance of these modifications is increasingly appreciated, their integration in the proteome remain unknown. Novel MS-based technologies identified 2,596 predominately unique sites in 1,319 mouse liver proteins under physiological conditions. Structural analysis localized the modifications in unique, evolutionary conserved protein segments, outside commonly annotated functional regions. Contrary to expectations, propensity for modification did not correlate with biophysical properties that regulate cysteine reactivity. However, the in vivo chemical reactivity is fine-tuned for specificity, demonstrated by the nominal complementation between the four modifications and quantitative proteomics that showed a reduction in S-nitrosylation is not correlated with increased S-glutathionylation. A comprehensive survey uncovered clustering of modifications within biologically related protein networks. The data provide the first evidence for the occurrence of distinct, endogenous protein networks that undergo redox signaling through specific cysteine modifications.