Differential expression of sterile alpha motif and leucine zipper-containing kinase AZK in cancers of the breast.

Breast cancer affects women at relatively high frequency (1). We mined published microarray datasets (2, 3) to determine in an unbiased fashion and at the systems level genes most differentially expressed in the primary tumors of patients with breast cancer. We report here significant differential expression of the gene encoding sterile alpha motif and leucine zipper-containing kinase AZK, ZAK, when comparing primary tumors of the breast to the tissue of origin, the normal breast. ZAK mRNA was present at significantly lower quantities in tumors of the breast as compared to normal breast tissue. Analysis of human survival data revealed that expression of ZAK in primary tumors of the breast was correlated with recurrence-free survival in patients with basal-like, luminal A and HER2+ subtype cancer, demonstrating a relationship between primary tumor expression of a differentially expressed gene and patient survival outcomes influenced by PAM50 molecular subtype. ZAK may be of relevance to initiation, maintenance or progression of cancers of the female breast.

Differential expression of sterile alpha motif and leucine zipper containing kinase AZK in human epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic cancer (1). We performed discovery of genes associated with epithelial ovarian cancer and of the high-grade serous ovarian cancer (HGSC) subtype, using published microarray data (2, 3) to compare global gene expression profiles of normal ovary or fallopian tube with that of primary tumors from women diagnosed with epithelial ovarian cancer or HGSC. We identified the gene encoding sterile alpha motif and leucine zipper containing kinase AZK, ZAK, as among the genes whose expression was most different in epithelial ovarian cancer as compared to the normal fallopian tube. ZAK expression was significantly lower in high-grade serous ovarian tumors relative to normal fallopian tube. ZAK expression correlated with progression-free survival in patients with ovarian cancer. These data indicate that expression of ZAK is perturbed in epithelial ovarian cancers broadly and in ovarian cancers of the HGSC subtype. ZAK may be relevant to pathways underlying ovarian cancer initiation (transformation) or progression.

ENHANCED GRAVITROPISM 2 encodes a STERILE ALPHA MOTIF–containing protein that controls root growth angle in barley and wheat

The root growth angle defines how roots grow toward the gravity vector and is among the most important determinants of root system architecture. It controls water uptake capacity, nutrient use efficiency, stress resilience, and, as a consequence, yield of crop plants. We demonstrated that the egt2 (enhanced gravitropism 2) mutant of barley exhibits steeper root growth of seminal and lateral roots and an auxin-independent higher responsiveness to gravity compared to wild-type plants. We cloned the EGT2 gene by a combination of bulked-segregant analysis and whole genome sequencing. Subsequent validation experiments by an independent CRISPR/Cas9 mutant allele demonstrated that egt2 encodes a STERILE ALPHA MOTIF domain–containing protein. In situ hybridization experiments illustrated that EGT2 is expressed from the root cap to the elongation zone. We demonstrated the evolutionary conserved role of EGT2 in root growth angle control between barley and wheat by knocking out the EGT2 orthologs in the A and B genomes of tetraploid durum wheat. By combining laser capture microdissection with RNA sequencing, we observed that seven expansin genes were transcriptionally down-regulated in the elongation zone. This is consistent with a role of EGT2 in this region of the root where the effect of gravity sensing is executed by differential cell elongation. Our findings suggest that EGT2 is an evolutionary conserved regulator of root growth angle in barley and wheat that could be a valuable target for root-based crop improvement strategies in cereals.

ENHANCED GRAVITROPISM 2 encodes a STERILE ALPHA MOTIVE containing protein that controls root growth angle in barley and wheat

The root growth angle defines how roots grow towards the gravity vector and is among the most important determinants of root system architecture. It controls water uptake capacity, nutrient use efficiency, stress resilience and as a consequence yield of crop plants. We demonstrated that the egt2 (enhanced gravitropism 2) mutant of barley exhibits steeper root growth of seminal and lateral roots and an auxin independent higher responsiveness to gravity compared to wild type plants. We cloned the EGT2 gene by a combination of bulked segregant analysis and whole genome sequencing. Subsequent validation experiments by an independent CRISPR/Cas9 mutant allele demonstrated that egt2 encodes a STERILE ALPHA MOTIF domain containing protein. In situ hybridization experiments illustrated that EGT2 is expressed from the root cap to the elongation zone. Subcellular localization experiments revealed that EGT2 localizes to the nucleus and cytoplasm. We demonstrated the evolutionary conserved role of EGT2 in root growth angle control between barley and wheat by knocking out the EGT2 orthologs in the A and B genomes of tetraploid durum wheat. By combining laser capture microdissection with RNA-seq, we observed that seven expansin genes were transcriptionally downregulated in the elongation zone. This is consistent with a role of EGT2 in this region of the root where the effect of gravity sensing is executed by differential cell elongation. Our findings suggest that EGT2 is an evolutionary conserved regulator of root growth angle in barley and wheat that could be a valuable target for root-based crop improvement strategies in cereals.Significance StatementTo date the potential of utilizing root traits in plant breeding remains largely untapped. In this study we cloned and characterized the ENHANCED GRAVITROPISM2 (EGT2) gene of barley that encodes a STERILE ALPHA MOTIF domain containing protein. We demonstrated that EGT2 is a key gene of root growth angle regulation in response to gravity which is conserved in barley and wheat and could be a promising target for crop improvement in cereals.

Phase separation by the polyhomeotic sterile alpha motif compartmentalizes Polycomb Group proteins and enhances their activity

Polycomb Group (PcG) proteins organize chromatin at multiple scales to regulate gene expression. A conserved Sterile Alpha Motif (SAM) in the Polycomb Repressive Complex 1 (PRC1) subunit Polyhomeotic (Ph) has been shown to play an important role in chromatin compaction and large-scale chromatin organization. Ph SAM forms helical head to tail polymers, and SAM-SAM interactions between chromatin-bound Ph/PRC1 are believed to compact chromatin and mediate long-range interactions. To understand the underlying mechanism, here we analyze the effects of Ph SAM on chromatin in vitro. We find that incubation of chromatin or DNA with a truncated Ph protein containing the SAM results in formation of concentrated, phase-separated condensates. Ph SAM-dependent condensates can recruit PRC1 from extracts and enhance PRC1 ubiquitin ligase activity towards histone H2A. We show that overexpression of Ph with an intact SAM increases ubiquitylated H2A in cells. Thus, SAM-induced phase separation, in the context of Ph, can mediate large-scale compaction of chromatin into biochemical compartments that facilitate histone modification.

Phase separation by the Sterile Alpha Motif of Polyhomeotic compartmentalizes Polycomb Group proteins and enhances their activity

Polycomb Group (PcG) proteins organize chromatin at multiple scales to regulate gene expression. A conserved Sterile Alpha Motif (SAM) in the Polycomb Repressive Complex 1 (PRC1) subunit Polyhomeotic (Ph) is important for chromatin compaction and large-scale chromatin organization. Like many SAMs, Ph SAM forms helical head to tail polymers, and SAM-SAM interactions between chromatin-bound Ph/PRC1 are believed to compact chromatin and mediate long-range interactions. To understand mechanistically how this occurs, we analyzed the effects of Ph SAM on chromatin in vitro. We find that incubation of chromatin or DNA with a truncated Ph protein containing the SAM results in formation of concentrated, phase-separated condensates. Condensate formation depends on Ph SAM, and is enhanced by but not strictly dependent on, its polymerization activity. Ph SAM-dependent condensates can recruit PRC1 from extracts and enhance PRC1 ubiquitin ligase activity towards histone H2A. Overexpression of Ph with an intact SAM increases ubiquitylated H2A in cells. Thus, phase separation is an activity of the SAM, which, in the context of Ph, can mediate large-scale compaction of chromatin into biochemical compartments that facilitate histone modification.