IndraLab
Statements
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"Here, there are comparable levels of EKLF at the promoter in both WT and Nan/+, but ectopic Nan-EKLF binding at a distal intron leads to higher pausing specifically in Nan/+ at that intronic site as well as the TSS ( xref and xref ), and this pattern is similar to Crtc1 , Pacs2 , and Mob2 of the hi-PI gene set ( xref - xref )."
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"The E339D mutation not only yields a variant with a more circumscribed binding specificity compared to wild type (WT), xref but also one that recognizes a novel, more degenerate target sequence uniquely recognized by Nan-KLF1. xref , xref The second observation is that, unlike the Nan mutant, mouse erythroid cells totally ablated in KLF1 do not enucleate, but instead stall at the orthochromatic erythroblast stage. xref Many of these cells are also bi-nucleated."
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"One of the most far reaching results from analysis of the Nan-KLF1 mutant was that the neomorphic expression pattern xref led to systemic effects that altered the hematologic properties of the mouse, including changes in levels of specific proteins and cytokines in the serum and feedback inhibition of erythropoiesis. xref In the present case, expression of the unique CDA genes is truly ectopic, as they do not overlap genes up-regulated in the KLF1-defective hydrops erythroid cell xref ( xref )."
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"IL17RB is critical for expression of IL8 ( CXCL8 ), a molecule that, if mis-expressed, could have systemic effects beyond the erythroid cell, particularly with respect to neutrophil activation and respiratory inflammation xref , xref (analogous to the misexpression of IFNβ in the Nan-KLF1 erythroid cell). xref We find that IL8 RNA expression levels are quite high in the CDA samples, but not detectable in the WT ( xref )."
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"Such aberrant activation of genes encoding secreted factors that exert a negative effect on erythropoiesis and iron use together with intrinsic hypomorphic effects of Nan-KLF1 within erythroid compartment contribute to exacerbation of the severity of neonatal anemia [ xref , xref , xref , xref ]."
sparser
"This is consistent with earlier findings that Nan-EKLF does not prefer binding to the cognate EKLF binding sequence with CAC (on the C-rich strand) due to the nature of the Nan mutation. xref , xref For peaks called exclusively in Nan/+ and not in WT (likely bound by Nan-EKLF), we found that, along with the altered preference in the middle nucleotide ( xref , denoted by star), there is also degeneracy at the adjacent nucleotide ( xref , denoted by circle) which is normally strictly G (on the G-rich strand) in WT. xref "
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"This is consistent with the known cooperativity of EKLF and Gata1 in transcription regulation during definitive erythropoiesis. xref Interestingly, GATA motifs are not co-enriched with EKLF peaks in Nan/+ ( xref ), suggesting that Nan-EKLF binds to regions of the genome independent of Gata1 binding."
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"Other ectopic genes such as Serpine1 and Fam159b have ectopic Nan-EKLF binding at the promoter, correlating with ectopic ATAC and CBP peaks at the TSS and correspondingly higher H3K27ac occupancy along the gene in Nan/+ ( xref and xref ), indicating that for Fam159b and Serpine1 , ectopic expression in Nan/+ is driven by the promoter."
sparser
"From a structural point of view, it is interesting that the conservative E-to-D change not only limits the recognition of the central nucleotide in the target DNA sequence (as noted previously xref , xref ), but also reduces the specificity of the overall 9-bp target recognition sequence from CCM CRC CCN to N CM YK C C Y N. Nevertheless, we find that Nan-EKLF DNA binding preferences from our data are consistent with earlier biochemical studies and previous determinations of Nan-EKLF target site preference (CCM NK C C Y N). xref , xref The central glutamate is conserved at that site within zinc-finger 2 in all KLF proteins across all species, xref - xref and X-ray studies of the highly similar KLF4 zinc fingers demonstrate that substitution of D alters the cognate recognition. xref , xref Further, the co-occurrence of Gata1 motifs in WT but not Nan-EKLF sites suggests that binding sites of lineage transcription factors have “clustered” during evolution to activate genes involved in a particular developmental process synchronously. xref - xref In addition, our studies demonstrate that the presence of a KLF protein with a change that significantly alters binding is detrimental and not biologically well tolerated within the cell, leading to a significantly altered genetic output even in the company of the WT."