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KLF1 mutation in human anemia
throblast and orthchromatic stages.46,47 Starting from the dataset of genes that are highest in the proerythroblast (‘cluster 16’ set in Figure 3B), we see that many of these genes continue to increase in expression during the normal erythroid lineage differentiation process (Figure 3C). Focusing first on the WT series, we see that the d11 set is most closely aligned with the polychromatic erythroblast expression pattern (Figure 3C). Many of these genes then decrease in expression upon further proliferation (d15), but most obviously after differentiation is established. The pattern for the CDA series is different in two signifi- cant ways (Figure 3C). 1) The expression of this gene set at d11 is quite low, consistent with its GSEA expression analysis. 2) Although the d15 set is converging on the polychromatic/orthochromatic erythroblast pattern, it remains minimally changed even after differentiation.
These data suggest that the CDA erythroid cell retains a significant residual early proliferation expression pattern, consistent with the erythroid hyperplasia seen in the CDA type IV patients’ bone marrow.4,5,12,13 Further supporting this, expression of early hematopoietic transcriptional markers such as GATA2 and RUNX1 remain high in the CDA samples (Figure 3D). We conclude that terminal dif- ferentiation is aberrant and does not proceed properly in the CDA erythroid cell; importantly, this follows from a combination of both the presence of KLF1-E325K and a hypomorphic level of total KLF1 RNA expression.
Ectopic expression of non-erythroid genes
The E325K change in KLF1-CDA is at a critical amino acid within the DNA recognition sequence. Based on structural arguments10,14 as well as the precedent from the Nan-KLF1 mutation at the same site (E339D),31,32 it is likely that the change in KLF1-CDA confers recognition of atyp- ical sites in the genome. This would lead to ectopic expression of genes that are normally not expressed in the erythroid cell. Substitution of a lysine for glutamate at amino acid (aa) 325 alters the middle residue of the critical “X,Y,Z” amino acids, which play determining roles in DNA target site recognition.48,49 Based on the most parsi- monious model, one may predict the K325 residue would now recognize guanine on the G-rich strand and alter the recognition sequence to 3’GGKGGGGGN5’. This would be a significant change, as a pyrimidine (T or C) is normal- ly present at the underlined site.
To identify these potential ectopic targets, we over- lapped data sets from a Venn analysis of all expressed genes (≥5 FPKM) that are exclusive to CDA (i.e. not expressed in WT) in proliferating d11, d15, and differenti- ating d5 samples. This yields a unique set of 184 genes (Figure 4A). Many of these are membrane proteins that are normally enriched in lymphoid, dendritic, myeloid, or monocyte cells. Perusal of the top 35 differentially expressed of these show that red cell character and identi- ty have been altered.
One of the most far reaching results from analysis of the Nan-KLF1 mutant was that the neomorphic expression pattern32 led to systemic effects that altered the hemato- logic properties of the mouse, including changes in levels of specific proteins and cytokines in the serum and feed- back inhibition of erythropoiesis.33 In the present case, expression of the unique CDA genes is truly ectopic, as they do not overlap genes up-regulated in the KLF1-defec- tive hydrops erythroid cell29 (Figure 4B). The extent of the level of misexpression of specific, normally non-erythroid
targets in the CDA cell is shown in Figure 4C. CCL13 is a chemokine implicated in inflammation, with potential respiratory issues. This connects it with LTC4S, which codes for leukotriene synthase, a gene normally expressed only in the lung whose product is also implicated in inflammation and respiration. PDPN codes for podoplas- min, also expressed in the lung but in addition implicated in aberrant platelet aggregation. IL17RB is a protein that binds to the IL17 receptor. These increases are not due to a global dysregulation, as expression of genes adjacent to those affected are not significantly changed (data not shown).
Of mechanistic interest, each of these genomic regions (CCL13, LTC4S, PDPN, IL17RB) contain multiple copies of the predicted novel recognition sequence that could potentially be recognized by KLF1-E325K (Figure 4D). In support of this idea, a recent study in the Siatecka lab, based on a binding site-selection strategy for CDA zinc fingers, demonstrates that each of these putative sites bind in vitro to CDA-KLF1, but not WT KLF1, as judged by gel shift assays (K Kulczynska et al., 2019, submitted manu- script).
IL17RB is critical for expression of IL8 (CXCL8), a mole- cule that, if mis-expressed, could have systemic effects beyond the erythroid cell, particularly with respect to neu- trophil activation and respiratory inflammation50,51 (analo- gous to the misexpression of IFNβ in the Nan-KLF1 ery- throid cell).33 We find that IL8 RNA expression levels are quite high in the CDA samples, but not detectable in the WT (Figure 4C). Consistent with the model, the CXCL8 genomic region does not contain potential ectopic binding sites for CDA-KLF1 (data not shown) and thus is likely indi- rectly activated by KLF1 through IL17RB.
We conclude that these and other ectopic targets are mis-expressed in the CDA erythroid cell by virtue of expression of KLF1-E325K and its action via its novel recognition site.
Discussion
Altered erythroid biology in the CDA patient cell
A mutation in KLF1, at E325K, is responsible for CDA type IV. Seven patients have been described so far, and they all share some similar phenotypic and clinical charac- teristics.4-13 However, changes of RNA expression within these patients’ erythroid cells has not been previously described. This is of interest as type IV is unique among CDA in that it is a transcription factor, rather than a struc- tural protein, that is mutated.1,3 We find a major alteration in the normal patterns of red cell gene expression, such that globin regulation, cell surface protein expression, membrane transport, cytokinesis, and iron utilization are all dramatically affected. These transcriptional effects go far in explaining the common patient phenotypes described in the literature.
However, a cautionary note must be acknowledged, stemming from the limitation that we could only analyze samples from one patient due to the rarity of the disease. The question is whether our documented changes could be due to inter-individual variation and/or whether we are comparing equivalent stages. This is a general question in the field that needs to be considered (e.g. as extensively discussed),52-54 particularly with respect to difficulties in finding the best way to compare patient/normal popula-
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