Bala Bharathi Burugula

The expression level of the low-density lipoprotein receptor ( LDLR ) gene is a critical determinant of circulating low-density lipoprotein concentrations and the corresponding risk of atherosclerotic cardiovascular disease. While the importance of sterol response elements in LDLR transcriptional regulation has been firmly established, the functional contribution of other potential cis -regulatory elements (CREs) remains poorly understood. In this study, we developed and applied a variety of high throughput functional genomic tools to systematically interrogate the noncoding space at the LDLR locus. These included saturation CRISPR nuclease, CRISPR activation, and CRISPR inhibition screening as well as a massively parallel reporter assay. With each approach, we observed robust discrimination of control sequences and significant effects for other regions with a previously unrecognized influence on LDLR expression. The CREs identified by each modality were overlapping but distinct, both in relation to each other and to those regions predicted to harbor CRE activity. Collectively, our findings clarify the functional landscape of the LDLR locus and highlight the complementary nature of different functional genomic approaches to CRE identification.

Lysosomes and peroxisomes are essential for cellular homeostasis, yet how their activities are coordinated remains poorly understood. Here, we identify peroxisome-derived ether lipids as key regulators of lysosomal function. A genome-wide CRISPR/Cas9 screen in LYSET-deficient mucolipidosis V cells revealed that disruption of ether lipid synthesis genes or peroxins markedly reduces lysosome accumulation and restores degradative capacity. Genetic or pharmacological inhibition of ether lipid synthesis enhanced lysosomal exocytosis and promoted the clearance of undigested material independently of mannose-6-phosphate trafficking. Conversely, supplementation with the ether lipid precursor hexadecylglycerol increased lysosome abundance, while reducing their degradative capacity. These findings uncover a peroxisome-lysosome metabolic axis, in which ether lipids act as bidirectional regulators of lysosomal number and function independently of the lysosomal master regulator TFEB. Our findings reveal how peroxisome-localized lipid metabolism modulates lysosomal homeostasis, and suggest potential new strategies to combat lysosomal and peroxisomal disorders.

Mutations in a diverse set of driver genes increase the fitness of haematopoietic stem cells (HSCs), leading to clonal haematopoiesis¹. These lesions are precursors for blood cancers^2,3,4,5,6, but the basis of their fitness advantage remains largely unknown, partly owing to a paucity of large cohorts in which the clonal expansion rate has been assessed by longitudinal sampling. Here, to circumvent this limitation, we developed a method to infer the expansion rate from data from a single time point. We applied this method to 5,071 people with clonal haematopoiesis. A genome-wide association study revealed that a common inherited polymorphism in the TCL1A promoter was associated with a slower expansion rate in clonal haematopoiesis overall, but the effect varied by driver gene. Those carrying this protective allele exhibited markedly reduced growth rates or prevalence of clones with driver mutations in TET2, ASXL1, SF3B1 and SRSF2, but this effect was not seen in clones with driver mutations in DNMT3A. TCL1A was not expressed in normal or DNMT3A-mutated HSCs, but the introduction of mutations in TET2 or ASXL1 led to the expression of TCL1A protein and the expansion of HSCs in vitro. The protective allele restricted TCL1A expression and expansion of mutant HSCs, as did experimental knockdown of TCL1A expression. Forced expression of TCL1A promoted the expansion of human HSCs in vitro and mouse HSCs in vivo. Our results indicate that the fitness advantage of several commonly mutated driver genes in clonal haematopoiesis may be mediated by TCL1A activation.

Frasier Syndrome (FS) is a rare Mendelian form of nephrotic syndrome caused by variants which disrupt the proper splicing of WT1 . This key transcription factor gene is alternatively spliced at exon 9 to produce two isoforms (“KTS+” and “KTS-”), which are normally expressed in the kidney at a ∼2:1 (KTS+:KTS-) ratio. FS results from variants that reduce this ratio by disrupting the splice donor of the KTS+ isoform. FS is extremely rare, and it is unclear whether any variants beyond the eight already known could cause FS. To prospectively identify other splicing-disruptive variants, we leveraged a massively parallel splicing assay. We tested every possible single nucleotide variant (n=519) in and around WT1 exon 9 for effects upon exon inclusion and KTS+/- ratio. Splice disruptive variants made up 11% of the tested point variants overall, and were tightly concentrated near the canonical acceptor and the KTS+/- alternate donors. Our map successfully identified all eight known FS or focal segmental glomerulosclerosis variants and 16 additional novel variants which were comparably disruptive to these known pathogenic variants. We also identified 19 variants that, conversely, increased the KTS+/KTS- ratio, of which two are observed in unrelated individuals with 46,XX ovotesticular disorder of sex development (46,XX OTDSD). This splicing effect map can serve as functional evidence to guide the clinical interpretation of newly observed variants in and around WT1 exon 9.

Two Letters to Blood address the risks of COVID-19 in populations with precursors of hematological disease. In the first article, Miller and colleagues report on whether clonal hematopoiesis of intermediate potential (CHIP) is associated with adverse outcomes with COVID-19, finding no association between CHIP and 28-day mortality while providing data indirectly linking IL-6 signaling and patient outcomes. In the second article, Ho and colleagues investigate the outcomes of patients with monoclonal gammopathy of undetermined significance (MGUS) with COVID-19, reporting that one-fourth had a severe infection and that on multivariable analysis, adverse outcomes are more likely if immunoparesis is present.

The mannose-6-phosphate (M6P) biosynthetic pathway for lysosome biogenesis has been studied for decades and is considered a well-understood topic. However, whether this pathway is regulated remains an open question. In a genome-wide CRISPR/Cas9 knockout screen, we discover TMEM251 as the first regulator of the M6P modification. Deleting TMEM251 causes mistargeting of most lysosomal enzymes due to their loss of M6P modification and accumulation of numerous undigested materials. We further demonstrate that TMEM251 localizes to the Golgi and is required for the cleavage and activity of GNPT, the enzyme that catalyzes M6P modification. In zebrafish, TMEM251 deletion leads to severe developmental defects including heart edema and skeletal dysplasia, which phenocopies Mucolipidosis Type II. Our discovery provides a mechanism for the newly discovered human disease caused by TMEM251 mutations. We name TMEM251 as GNPTAB cleavage and activity factor (GCAF) and its related disease as Mucolipidosis Type V.

The mannose-6-phosphate (M6P) biosynthetic pathway for lysosome biogenesis has been studied for decades and is considered a well-understood topic. However, whether this pathway is regulated remains an open question. In a genome-wide CRISPR/Cas9 knockout screen, we discovered TMEM251 as the first regulator of the M6P modification. Deleting TMEM251 caused mistargeting of most lysosomal enzymes due to their loss of M6P modification and accumulation of numerous undigested materials. We further demonstrated that TMEM251 localizes to the Golgi and is required for the cleavage and activation of GNPT, the enzyme that catalyzes M6P modification. In zebrafish, TMEM251 deletion leads to severe developmental defects including heart edema and skeletal dysplasia, which phenocopies Mucolipidosis Type II. Our discovery provides a mechanism for the newly discovered human disease caused by TMEM251 mutations. We name TMEM251 as GNPTAB cleavage and activation factor (GCAF) and its related disease as Mucolipidosis Type V.

The lack of functional evidence for the majority of missense variants limits their clinical interpretability, and poses a key barrier to the broad utility of carrier screening. In Lynch Syndrome (LS), one of the most highly prevalent cancer syndromes, nearly 90% of clinically observed missense variants are deemed “variants of uncertain significance” (VUS). To systematically resolve their functional status, we performed a massively parallel screen in human cells to identify loss-of-function missense variants in the key DNA mismatch repair factor MSH2 . The resulting functional effect map is substantially complete, covering 94% of the 17,746 possible variants, and is highly concordant (96%) with existing functional data and expert clinicians’ interpretations. The large majority (89%) of missense variants were functionally neutral, perhaps unexpectedly in light of its evolutionary conservation. These data provide ready-to-use functional evidence to resolve the ∼1,300 extant missense VUSs in MSH2 , and may facilitate the prospective classification of newly discovered variants in the clinic.

Substance P neuropeptide and its receptor, neurokinin-1 receptor (NK1R), are reported to present on the ocular surface. In this study, mice lacking functional NK1R exhibited an excessive desquamation of apical corneal epithelial cells in association with an increased epithelial cell proliferation and increased epithelial cell density, but decreased epithelial cell size. The lack of NK1R also resulted in decreased density of corneal nerves, corneal epithelial dendritic cells (DCs), and a reduced volume of basal tears. Interestingly, massive accumulation of CD11c+CD11b+ conventional DCs was noted in the bulbar conjunctiva and near the limbal area of corneas from NK1R−/− mice. After ocular HSV-1 infection, the number of conventional DCs and neutrophils infiltrating the infected corneas was significantly higher in NK1R−/− than C57BL/6J mice. This was associated with an increased viral load in infected corneas of NK1R−/− mice. As a result, the number of IFN-γ–secreting virus-specific CD4 T cells in the draining lymph nodes of NK1R−/− mice was much higher than in infected C57BL/6J mice. An increased number of CD4 T cells and mature neutrophils (CD11b+Ly6ghigh) in the inflamed corneas of NK1R−/− mice was associated with an early development of severe herpes stromal keratitis. Collectively, our results show that the altered corneal biology of uninfected NK1R−/− mice along with an enhanced immunological response after ocular HSV-1 infection causes an early development of herpes stromal keratitis in NK1R−/− mice.

Spt6 is a multifunctional histone chaperone involved in the maintenance of chromatin structure during elongation by RNA polymerase II (Pol II). Spt6 has a tandem SH2 (tSH2) domain within its C terminus that recognizes Pol II C-terminal domain (CTD) peptides phosphorylated on Ser2, Ser5, or Try1 in vitro. Deleting the tSH2 domain, however, only has a partial effect on Spt6 occupancy in vivo, suggesting that more complex mechanisms are involved in the Spt6 recruitment. Our results show that the Ser2 kinases Bur1 and Ctk1, but not the Ser5 kinase Kin28, cooperate in recruiting Spt6, genome-wide. Interestingly, the Ser2 kinases promote the association of Spt6 in early transcribed regions and not toward the 3′ ends of genes, where phosphorylated Ser2 reaches its maximum level. In addition, our results uncover an unexpected role for histone deacetylases (Rpd3 and Hos2) in promoting Spt6 interaction with elongating Pol II. Finally, our data suggest that phosphorylation of the Pol II CTD on Tyr1 promotes the association of Spt6 with the 3′ ends of transcribed genes, independently of Ser2 phosphorylation. Collectively, our results show that a complex network of interactions, involving the Spt6 tSH2 domain, CTD phosphorylation, and histone deacetylases, coordinate the recruitment of Spt6 to transcribed genes in vivo.