Direct Recruitment of ERK Cascade Components to Inducible Genes Is Regulated by Heterogeneous Nuclear Ribonucleoprotein (hnRNP) K

Abstract

Components of the ERK cascade are recruited to genes, but it remains unknown how they are regulated at these sites. The RNA-binding protein heterogeneous nuclear ribonucleoprotein (hnRNP) K interacts with kinases and is found along genes including the mitogen-inducible early response gene EGR-1. Here, we used chromatin immunoprecipitations to study co-recruitment of hnRNP K and ERK cascade activity along the EGR-1 gene. These measurements revealed that the spatiotemporal binding patterns of ERK cascade transducers (GRB2, SOS, B-Raf, MEK, and ERK) at the EGR-1 locus resemble both hnRNP K and RNA polymerase II (Pol II). Inhibition of EGR-1 transcription with either serum-responsive factor knockdown or 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole altered recruitment of all of the above ERK cascade components along this locus that mirrored the changes in Pol II and hnRNP K profiles. siRNA knockdown of hnRNP K decreased the levels of active MEK and ERK at the EGR-1, changes associated with decreased levels of elongating pre-mRNA and less efficient splicing. The hnRNP K dependence and pattern of ERK cascade activation at the c-MYC locus were different from at EGR-1. Ribonucleoprotein immunoprecipitations revealed that hnRNP K was associated with the EGR-1 but not c-MYC mRNAs. These data suggest a model where Pol II transcription-driven recruitment of hnRNP K along the EGR-1 locus compartmentalizes activation of the ERK cascade at these genes, events that regulate synthesis of mature mRNA.

Heterogeneous Nuclear Ribonucleoprotein (HnRNP) K Genome-wide Binding Survey Reveals Its Role in Regulating 3′-End RNA Processing and Transcription Termination at the Early Growth Response 1 (EGR1) Gene through XRN2 Exonuclease*

“…We have recently found that hnRNPK is recruited along several inducible genes with a pattern mirroring the Pol2 complex, an event that regulates the activity of the MAPK pathway components at these sites (11). To gain more insight about the role of its inducible chromatin binding we compared ChIP-Seq hnRNPK analyses of the human colon carcinoma HCT-116 line in quiescent and proliferative states.…”

“…Cells-HCT116 WT human colon carcinoma cells lines were grown in plastic 6-well plates in McCoy's media supplemented with 10% FBS (11). For sequencing, chromatin was isolated from 24-h serum-starved cells (0.5% FBS) and cells were stimulated for 30 min with 10% FBS.…”

“…Cells were treated 24 h later in McCoy's media supplemented with 10% FBS for the given time points. Cells were harvested, lysates were prepared and used in RIP assay using rabbit IgG (I-1000, Vector Labs), hnRNPK #54, and XRN2 (ab72181) antibody as described (11).…”

“…HnRNPK has been shown to directly bind and regulate expression from multiple promoters including c-Myc (5), c-Src (6), thymidine kinase 1 (7), androgen receptor (8), eukaryotic translation initiation factor 4E (9), and p53 target genes (10). Using chromatin immunoprecipitation (ChIP) assays we have recently shown that hnRNPK recruitment along genes mirrors occupancy of RNA polymerase II (Pol2) suggesting it may also play a role beyond the initiation step of transcription (11). The advent of the next generation sequencing technique together with the ChIP method (ChIP-Seq) allows to study protein-DNA interactions genome-wide at single nucleotide resolutions (12).…”

Heterogeneous Nuclear Ribonucleoprotein K (hnRNP-K) Promotes Tumor Metastasis by Induction of Genes Involved in Extracellular Matrix, Cell Movement, and Angiogenesis

“…Furthermore, NIH 3T3 cells treated with U0126 lost hnRNP-Kinduced up-regulation of MMP-3, demonstrating that the hnRNP-K-regulated cell migration is by the Ras/MEK/ERK-MMP-3 pathway. Mikula et al (46) used chromatin immunoprecipitations to study co-recruitment of hnRNP-K and ERK cascade activity along with the Egr-1 gene and found that the spatiotemporal binding patterns of ERK cascade transducers (GRB2, SOS, B-Raf, MEK, and ERK) at the EGR-1 locus resemble both hnRNP-K and RNA polymerase II. Furthermore, knockdown of hnRNP-K by siRNA decreased the level of active MEK and ERK suggesting that hnRNP-K regulates MEK and ERK as seen in the present study.…”