Chromatin enrichment for proteomics in plants (ChEP-P) implicates the histone reader ALFIN-LIKE 6 in jasmonate signalling

Background Covalent modifications of core histones govern downstream DNA-templated processes such as transcription by altering chromatin structure and function. Previously, we reported that the plant homeodomain protein ALFIN-LIKE 6 (AL6), a bona fide histone reader that preferentially binds trimethylated lysin 4 on histone 3 (H3K4me3), is critical for recalibration of cellular phosphate (Pi) homeostasis and root hair elongation under Pi-deficient conditions. Results Here, we demonstrate that AL6 is also involved in the response of Arabidopsis seedlings to jasmonic acid (JA) during skotomorphogenesis, possibly by modulating chromatin dynamics that affect the transcriptional regulation of JA-responsive genes. Dark-grown al6 seedlings showed a compromised reduction in hypocotyl elongation upon exogenously supplied JA, a response that was calibrated by the availability of Pi in the growth medium. A comparison of protein profiles between wild-type and al6 mutant seedlings using a quantitative Chromatin Enrichment for Proteomics (ChEP) approach, that we modified for plant tissue and designated ChEP-P (ChEP in Plants), yielded a comprehensive suite of chromatin-associated proteins and candidates that may be causative for the mutant phenotype. Conclusions Altered abundance of proteins involved in chromatin organization in al6 seedlings suggests a role of AL6 in coordinating the deposition of histone variants upon perception of internal or environmental stimuli. Our study shows that ChEP-P is well suited to gain holistic insights into chromatin-related processes in plants. Data are available via ProteomeXchange with identifier PXD026541.

Chromatin Enrichment for Proteomics in Plants (ChEP-P) Implicates the Histone Reader ALFIN-LIKE 6 in Jasmonate Signalling

BackgroundCovalent modifications of core histonesgoverndownstream DNA-templated processes such as transcription by altering chromatin structure and function. Previously, we reported that the plant homeodomain protein ALFIN-LIKE6 (AL6), a bona fide histone reader that preferentially binds trimethylated lysin 4 on histone 3 (H3K4me3), is critical for recalibration of cellular phosphate (Pi) homeostasis and root hair elongation under Pi-deficient conditions. ResultsHere, we demonstrate that AL6 is also involved in the response of Arabidopsis seedlings to jasmonic acid (JA) during skotomorphogenesis, possibly by modulating chromatin dynamics that affect the transcriptional regulation of JA-responsivegenes. Dark-grown al6 seedlings showed a compromised reduction in hypocotyl elongation upon exogenously supplied JA, a response that was calibrated by the availability of Pi in the growth medium. A comparison of protein profiles between wild-type and al6 mutant seedlings using a quantitative Chromatin Enrichment for Proteomics (ChEP) approach,that we modified for plant tissue and designated ChEP-P (ChEP in Plants), yielded a comprehensive suite of chromatin-associated proteins and candidates that may be causative for the mutant phenotype. ConclusionsAltered abundance of proteins involved in chromatin organization in al6 seedlings suggests a role of AL6 in coordinating the deposition of histone variants upon perception of internal or environmental stimuli. Our study shows that ChEP-P is well suited to gain holistic insights into chromatin-related processes in plants. Data are available via ProteomeXchange with identifier PXD026541.

Synergistic roles of homeodomain proteins UNC-62 homothorax and MLS-2 HMX/NKX in the specification of olfactory neurons in Caenorhabditis elegans

General identity of the Caenorhabditis elegans AWC olfactory neuron pair is specified by the OTX/OTD transcription factor CEH-36 and the HMG-box transcription factor SOX-2, followed by asymmetrical differentiation of the pair into two distinct subtypes, default AWCOFF and induced AWCON, through a stochastic signaling event. The HMX/NKX transcription factor MLS-2 regulates the expression of ceh-36 to specify general AWC identity. However, general AWC identity is lost in only one of the two AWC cells in the majority of mls-2 null mutants displaying defective general AWC identity, suggesting that additional transcription factors have a partially overlapping role with MLS-2 in the specification of general AWC identity. Here we identify a role of unc-62, encoding a homothorax/Meis/TALE homeodomain protein, in the specification of general AWC identity. As in mls-2 null mutants, unc-62 null mutants showed an incomplete penetrance in loss of general AWC identity. However, unc-62; mls-2 double mutants display a nearly complete penetrance of identity loss in both AWC cells. Thus, unc-62 and mls-2 have a partially overlapping function in the specification of general AWC identity. Furthermore, our genetic results suggest that mls-2 and unc-62 act cell autonomously in promoting the AWCON subtype. Together, our findings reveal the sequential roles of the unc-62 and mls-2 pair in AWC development, specification of general AWC identity in early embryogenesis and asymmetric differentiation of AWC subtypes in late embryogenesis.

VPB1 Encoding BELL-like Homeodomain Protein Is Involved in Rice Panicle Architecture

Inflorescence architecture in rice (Oryza sativa) is mainly determined by spikelets and the branch arrangement. Primary branches initiate from inflorescence meristem in a spiral phyllotaxic manner, and further develop into the panicle branches. The branching patterns contribute largely to rice production. In this study, we characterized a rice verticillate primary branch 1(vpb1) mutant, which exhibited a clustered primary branches phenotype. Gene isolation revealed that VPB1 was a allele of RI, that it encoded a BELL-like homeodomain (BLH) protein. VPB1 gene preferentially expressed in the inflorescence and branch meristems. The arrangement of primary branch meristems was disturbed in the vpb1 mutant. Transcriptome analysis further revealed that VPB1 affected the expression of some genes involved in inflorescence meristem identity and hormone signaling pathways. In addition, the differentially expressed gene (DEG) promoter analysis showed that OsBOPs involved in boundary organ initiation were potential target genes of VPB1 protein. Electrophoretic mobility shift assay (EMSA) and dual-luciferase reporter system further verified that VPB1 protein bound to the promoter of OsBOP1 gene. Overall, our findings demonstrate that VPB1 controls inflorescence architecture by regulating the expression of genes involved in meristem maintenance and hormone pathways and by interacting with OsBOP genes.

BEL1-like Homeodomain Protein BLH6a Is a Negative Regulator of CAl5H2 in Sinapyl Alcohol Monolignol Biosynthesis in Poplar

Lignin is one of the major components of xylem cell walls in tree stems. The lignin in the wood of most flowering plants (dicotyledonous angiosperms) is typically polymerized from three monolignol precursors, coniferyl alcohol, sinapyl alcohol, and p-coumaroyl alcohol, resulting in guaiacyl (G), syringyl (S), and hydroxyphenyl (H) subunits, respectively. In this study, we focus on the transcriptional regulation of a coniferaldehyde 5-hydroxylase (CAld5H2) gene, which encodes a key enzyme for sinapyl alcohol biosynthesis. We carried out a yeast one-hybrid (Y1H) screen to identify candidate upstream transcription factors (TFs) regulating CAld5H2. We obtained 12 upstream TFs as potential regulators of CAld5H2. One of these TF genes, BLH6a, encodes a BEL1-like homeodomain (BLH) protein and negatively regulated the CAld5H2 promoter activity. The direct regulation of CAld5H2 promoter by BLH6a was supported by chromatin immunoprecipitation–quantitative polymerase chain reaction (ChIP–qPCR) and dominant repression of BLH6a in transgenic plants. Luciferase complementation imaging analyses showed extensive protein–protein interactions among these 12 TFs. We propose that BLH6a is a negative regulator of CAld5H2, which acts through combinatorial regulation of multiple TFs for sinapyl alcohol (S monolignol) biosynthesis in poplar.

Chromatin enrichment for Proteomics in Plants (ChEP-P) implicates the histone reader ALFIN-LIKE 6 in jasmonate signalling

Covalent modifications of core histones govern downstream DNA-templated processes such as transcription by altering chromatin structure and function. Previously, we reported that the plant homeodomain protein ALFIN-LIKE 6 (AL6), a bona fide histone reader that preferentially binds trimethylated lysin 4 on histone 3 (H3K4me3), is critical for recalibration of cellular phosphate (Pi) homeostasis and root hair elongation under Pi-deficient conditions. Here, we demonstrate that AL6 is also involved in the response of Arabidopsis seedlings to jasmonic acid (JA) during skotomorphogenesis, possibly by modulating chromatin dynamics that affect the transcriptional regulation of JA-responsive genes. Dark-grown al6 seedlings showed a compromised reduction in hypocotyl elongation upon exogenously supplied JA, a response that was calibrated by the availability of Pi in the growth medium. A comparison of protein profiles between wild-type and al6 mutant seedlings using a quantitative Chromatin Enrichment for Proteomics (ChEP) approach, that we modified for plant tissue and designated ChEP-P (ChEP in Plants), yielded a comprehensive suite of chromatin-associated proteins and candidates that may be causative for the mutant phenotype. Altered abundance of proteins involved in chromatin organization in al6 seedlings suggests a role of AL6 in coordinating the deposition of histone variants upon perception of internal or environmental stimuli.

Identification and Characterization of Alternatively Spliced Transcript Isoforms of IRX4 in Prostate Cancer

Alternative splicing (AS) is tightly regulated to maintain genomic stability in humans. However, tumor growth, metastasis and therapy resistance benefit from aberrant RNA splicing. Iroquois-class homeodomain protein 4 (IRX4) is a TALE homeobox transcription factor which has been implicated in prostate cancer (PCa) as a tumor suppressor through genome-wide association studies (GWAS) and functional follow-up studies. In the current study, we characterized 12 IRX4 transcripts in PCa cell lines, including seven novel transcripts by RT-PCR and sequencing. They demonstrate unique expression profiles between androgen-responsive and nonresponsive cell lines. These transcripts were significantly overexpressed in PCa cell lines and the cancer genome atlas program (TCGA) PCa clinical specimens, suggesting their probable involvement in PCa progression. Moreover, a PCa risk-associated SNP rs12653946 genotype GG was corelated with lower IRX4 transcript levels. Using mass spectrometry analysis, we identified two IRX4 protein isoforms (54.4 kDa, 57 kDa) comprising all the functional domains and two novel isoforms (40 kDa, 8.7 kDa) lacking functional domains. These IRX4 isoforms might induce distinct functional programming that could contribute to PCa hallmarks, thus providing novel insights into diagnostic, prognostic and therapeutic significance in PCa management.

Surface residues and non-additive interactions stabilize a consensus homeodomain protein

Despite the widely reported success of consensus design in producing highly stabilized proteins, little is known about the physical mechanisms underlying this stabilization. Here we explore the potential sources of stabilization by performing a systematic analysis of the 29 substitutions that we previously found to collectively stabilize a consensus homeodomain compared to an extant homeodomain. By separately introducing groups of consensus substitutions that alter or preserve charge state, occur at varying degrees of residue burial, and occur at positions of varying degrees of conservation, we determine the extent to which these three features contribute to the consensus stability enhancement. Surprisingly, we find that the largest total contribution to stability comes from consensus substitutions on the protein surface and that the largest per-substitution contributions come from substitutions that maintain charge state, suggesting that although consensus proteins are often enriched in charged residues, consensus stabilization does not result primarily from charge-charge interactions. Although consensus substitutions at strongly conserved positions also contribute disproportionately to stabilization, significant stabilization is also contributed from substitutions at weakly conserved positions. Furthermore, we find that identical consensus substitutions show larger stabilizing effects when introduced into the consensus background than when introduced into an extant homeodomain, indicating that synergistic, stabilizing interactions among the consensus residues contribute to consensus stability enhancement of the homeodomain.