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Zinc Fingers in Biological Systems

Lino Cardoz
From the very beginning biologist marvelled at cellular complexities that are found in nature, and were awe struck by the very nature of genetic information that was present within each living cell. Hence they began understanding the mechanism by which the cellular machinery would regulate and manipulate this genetic information at a molecular level [1]. With the aid of some cutting edge technology, X-ray crystallography, along with NMR and many other physical and chemical methods major breakthroughs were made in the late 1970 in studying protein-DNA interactions. The first X-ray crystallographic study of a protein–DNAcomplex was reported in 1984, by the end of the 20th century 240 structures were documented and the number has exploded ever since [2]. Protein–DNAcomplexs were grouped in classes like polymerases, transcription factors, nucleases and other enzymes and structural proteins based on their functions [3]. Zinc finger was one such protein-DNAcomplex which was initially discovered in the transcription factor TFIIIA from Xenopus oocytes [3]. The ZNF domain is found abundantly in eukaryotes (in Humans alone 300 and 700 human genes encode zinc finger-containing proteins (nearly 1% of the human genome), practically absent in bacteria with some exceptions as in plant pathogens, scarcely in archaea and their viruses. [3]
It was rather unusual to have found a ninefold repeating pattern of amino acids sequence in Transcriptional Factor IIIA. This ninefold pattern consisted of conserved cysteine, histidine, and hydrophobic residues which were arranged in fashion: -X-Cys-X2-5-Cys-X3–-X5–-X2-His-X2-5-His, where X represents any amino acid, hydrophobic residues and His Histidine [1]. With this observation it was proposed that the classical ZNF motif would consists of a short of 30amino acid residue motif, that would form an independent, DNA binding minidomain folded around a central zinc ion with tetrahedral arrangements of cysteine and histidine metal legends. These proposals were then confirmed by EXAFS (Extended x-ray absorption fine structure) analysis [1]. The zinc finger proteins typically contain several fingers that make contacts with the DNA and each finger folds into a compact domain with a single zinc ion that sandwiched between the two-stranded antiparallel sheet and the helix [2].
The C2H2 type zinc fingers are the most commonly DNA-binding motifs found in humans and genomes of most multicellular organisms. Its motif are made up of 23-26 residue consensus sequence that contain two conserved cysteine (C) and two conserved histidine (H) residues, whose side chains are bound to Zn2 ion on the other hand the C4 type zinc finger is found in approximately 50 human transcriptional factors has 4cysteine residues that bind to the zinc ion [4].
Only the helix of the zinc finger interacts with the DNA. The first x ray crystal structure that served as a prototype of the zinc finger family was Zif 268, a 90 amino acid portion of a mouse transcription factor. This protein comprises three zinc fingers where one side of the helix is inserted into the DNAmajor groove at an angle of 45 with respect to the base-pair plain and binding of the successive fingers causes the protein to wrap around DNA [5]. The contact to the primary DNA strand (5’-GCGTGGGCGT-3’) is made through the 1, 3 and 6 residue preceding the helix of the protein and the residue 2 binds to complimentary strand. Although these contacts mainly occur due to hydrogen bonds, van der Waals interactions and phosphate contact also contribute to the latter [5].
Zinc fingers domain containing proteins are “Wonder Molecules” as they perform some extraordinarily diverse functions in cellular processes such as DNA recognition, RNA packaging, transcriptional activation, regulation of apoptosis and metabolism, protein folding and assembly, and lipid binding [6,7]. Proteins from GATA-1 family found across a wide range of species from fungi to humans, play a role in regulating transcription with the help of four conserved cysteine residues and the ability to recognize the GATA DNA base sequence [1], yeast RNA polymerase II having six zinc-binding proteins also helps regulate transcription [7]. The Arf-GAP (ADP-ribosylation factor GTPase-activating proteins) has a GATA type zinc binding motif that helps it to regulate membrane trafficking and to mediate binding to phosphoinositides along with the pleckstrin homology (PH) domain [7]. The retroviral nucleocapsid (NC) protein from HIV-1 having 2 zinc ‘knuckles’ plays a major role in the recognition and packaging of the retroviral genome. The Fpg protein of Escherichia coli weighs 30.2 kDa and has C2H2zinc finger motifs. It shows DNA glycosylase, abasic site nicking, and deoxyribose excising activities and serves as a DNA repair enzyme by excising a variety of modified bases in the DNA [8]. Arabidopsis thaliana LSD1 (AtLSD1) contains three LSD1-type zinc finger motifs, which are involved in the protein–protein interaction. LSD1 plays a major role in negative regulation of plant programmed cell death (PCD) [9]. Zinc fingers are also known to regulate resistance mechanism for various biotic and abiotic stresses conditions plants (rice, wheat, tobacco, sunflower, tomato, barley, flax, and potato) faces, and being actively involved in their development and growth [10]. In recent years genome engineering in model organism such as Drosophila melanogaster,Arabidopsis thaliana, zebrafish and rats have been carried out using the application of the same core technology ie targeted genome cleavage by engineered, sequence-specific zinc finger nucleases followed by gene modification during subsequent repair [11].Soybean GmPHD-Type transcription regulators which contain zinc finger motifs improved drought, salt, cold tolerance and ABA treatments in transgenic Arabidopsis Plants [12]. A broader range of approaches will be available in the future as we uncover more of nature’s secrets to tackle the experimental challenges that limit us, zinc fingers will play a key role and stand as strong contenders in world of molecular editing.
Kiug, A., Schwabe, J. W. R. Zinc fingers. FASEBJ. 9, 597-604 (1995).
Yong Xiong and Muttaiya Sundaralingam. (2012) Protein–Nucleic Acid Interaction: Major Groove Recognition Determinants. Encyclopedia of life sciences /

Regulation of Human Haematopoietic Stem Cell Self-renewal

Regulation of human haematopoietic stem cell self-renewal by the microenvironment’s control of retinoic acid signalling
Ghiaura et al. (2013) PNAS 110 no. 40 pp 16121–16126
Constant blood cell production is dependent on perpetual reconstitution of the hematopoietic system via a series of lineage-restricted intermediates. This remarkable cell renewal process is supported by a rare and small subpopulation of bone marrow cells called hematopoietic stem cells (HSCs) (Notta et al. 2011). During differentiation, HSCs, after series of intermediates, gives rise to various blood cells of the myeloid and lymphoid lineages. It is important that these stem cells do not deplete as many systems are maintained by them hence the unique property termed stemness. Stemness is the unique ability of both embryonic and adult tissue stem cells to self-renew indefinitely and give rise to multiple cell lineages (Wong et al. 2008). Stem cells have a unique expression profile, and as such, they are identified by cell surface markers. Stem cell markers are genes and their protein products used to isolate and identify various types of stem cells (Pazhanisamy, 2013). A well-known HSC marker is aldehyde dehydrogenase (ALDH) also known as retinaldehyde dehydrogenases. Specifically, ALDH1 is a marker for stemness in both the haematopoietic system and other tissue since it is highly expressed by HSC but decreases as they differentiate. Although the precise function of ALDH1 in stem cell regeneration is unclear, it is known that ALDH1 is involved in the biosynthesis of retinoic acid (RA) as it catalyses the oxidation of retinaldehyde to RA, which binds to its receptor RA receptor-α (RARα) in the nucleus to drive transcription of genes (fig. 1). Thus, this led to the suggestion that RA signalling could be important is determining the fate of HSCs. Previous studies by Chute et al (2006) then showed that inhibition of ALDH1 and consequently de novo synthesis of RA not only delayed the differentiation of human HSCs but expanded the well-characterized CD34 CD38− hematopoietic stem and progenitor cells (HSPCs) from the umbilical cord blood. These cells were also capable of long-term engraftment of immunodeficient mice. Another study showed that RA in the Sertoli cells in the gonadal microenvironment embryonic mesonephroi was inactivated by RA through expression of CYP26B1 (in its stroma), thus, ultimately leading to spermatogenesis.
Although it is known that HSCs reside in a highly complex microenvironment that assures their survival, self-renewal, and differentiation; the microenvironment’s mechanisms responsible for maintaining HSC homeostasis remain unclear. Other studies have shown that HoxB4, Bmi1, Wnt/β-catenin, Notch, and aryl hydrocarbon receptor can promote expansion of umbilical cord blood hematopoietic cells. Consequently, this study was aimed at investigating the precise mechanism for the self-renewal of adult human bone marrow-derived HSCs using in vitro and in vivo assays.

Fig 1. Retinoic Acid Synthesis and Signalling. In an RA-generating tissue, retinol is oxidized to retinaldehyde by alcohol dehydrogenase (ADH) and retinaldehyde is oxidized to RA by retinaldehyde dehydrogenase (RALDH) also known as aldehyde dehydrogenase (ALDH). RA is then released and taken up by surrounding cells. RA non-target cells that express cytochrome P450 (CYP26) initiate the further oxidation of RA for degradation. Some RA target cells express cellular RA-binding protein (CRABP) that facilitates uptake of RA and transport to the nucleus where RA binds the RA receptor (RAR). The ternary complex of ligand-bound RAR with RXR and a retinoic acid response element (RARE) regulates transcription of RA target genes by altering the binding of corepressors and coactivators.
Relative expression of RAR pathway genes in CD34 CD38− and CD34 CD38 cells
It is generally accepted that at all HSC express the CD34 CD38– immunophenotype. This immunophenotype are a rare and primitive subpopulation of progenitor cells present in both foetal and adult bone marrow. These cells are functionally distinguished from the CD34 CD38 population by sustained clonogenicity. They differentiate to generate colony-forming unit-cells (CFU-Cs) following culture in the presence of growth factors (Hao et al. 1995); however, less is known about factors that govern the self-renewal of HSPCs and thus they are rapidly exhausted (Dahlberg et al. 2011).
To identify the pathway responsible for HSC fate, Ghiaur et al (2013) procured Bone marrow harvested from five normal donors. Bone marrow comprises of progenitors of skeletal and hematopoietic tissue; and adipocytes surrounded by blood vessels (Travlos, 2006). Thus, the cell subsets of interest were isolated and CD34-positive cells were then selected from mononuclear cells using magnetic beads and column. Viable CD34 cells were sorted by labelling with monoclonal fluorescein isothiocyanat (FITC)-conjugated anti-CD34 and allophycocyanin (APC)-conjugated anti-CD38 and sorted by using fluorescence activated cell sorter (FACS). Ghiaur et al (2013) then obtained a genome-wide exon microarray data on the primitive human bone marrow-derived CD34 CD38– and CD34 CD38 cells (more differentiated). They observed that the RA pathway was one of the top pathways that showed coordinated inactivation in CD34 CD38– compared with the CD34 CD38 population. They also found that in CD34 CD38− cells, the transcriptional level of ALDH1 and RARα was upregulated. This was identified by the Ingenuity Pathway Analysis (IPA) Upstream Regulator module. However, the up-regulation of these early components of RA signalling, did not lead to a concomitant activation of the downstream pathway in the primitive CD34 CD38− cells.
Effect of RA signalling in bone marrow-derived cells
The previous results thus led to the evaluation of the effect of suppression of the downstream RA signalling in maintaining the CD34 CD38– phenotype. CD34 CD38− cells were cultured in serum-free media containing thrombopoietin, stem- cell factor and Flt3-ligand (TSF) with and without the pan-RAR inhibitor AGN194310 (AGN). Ghiaur et al found that culture of the CD34 CD38− cells with TSF generated CD34 CD38− cells and the primitive CAFCW8 but by days 21–28 of culture, both cells declined. Also, CFU-Cs were rapidly generated but reached a plateau at approximately day 21. Interestingly, they observed that inhibition of RA signalling using AGN resulted in a relative lag in generation of CFU-Cs during the first 7–14 days compared with control cultures. However, by day 28, the number of CFU-Cs expanded beyond the control cultures, resulting in a statistically higher overall output of CFU-C (Fig. 2A). They also found that by adding a RAR agonist, all-trans RA, the effects of AGN was reversed (fig 2B). Thus, they concluded that suppression of RA signalling may be crucial in promoting self-renewal of bone marrow-derived primitive HSCs.

Fig. 2. Effects of retinoic acid (RA) signalling on the committed hematopoietic progenitor cells-CFU-Cs. Co-culture of CD34 CD38− cells with thrombopoietin, stem-cell factor, and Flt3-ligand (TSF) alone or with the addition of 1 μM AGN194310 (AGN) and/ 0.1 μM all-trans RA (ATRA). A) Colony-forming unit cells CFU-Cs reached a plateau at approximately day 21 in cultures with TSF alone. In the presence of AGN, there was a significant expansion of CFU-C at day 28 of liquid culture [61.7 ± 8.1 (AGN) vs. 40.1 ± 7 (TSF)].b) ATRA significantly reduced CAFCW8 recovery.
Among the distinctive characteristics of hematopoietic stem cells (HSCs) is their ability to differentiate and repopulate irradiated/immunodeficient recipients. To assess the level of human engraftment, Ghiaur et al transplanted CD34 CD38– cells in limiting dilution into sublethally irradiated (225 cGy) NOD/SCID-IL2Rγ−/−(NSG) mice before and after in vitro culture; and engraftment was analysed 18–20 weeks after transplantation. They observed that overall, CD34 CD38− cells from AGN–treated culture showed higher human engraftment in NSG mice whereas CD34 CD38− cells cultured in the presence of TSF alone demonstrated a rapid decline of engraftment (fig 3).

Fig 3. Effect of inhibition of RA signalling on NSG engraftment. Mice transplanted with cells treated with AGN generally had higher level of engraftment compared with control, especially at day 7.
Bone Marrow Stromal Contributes to the Maintenance of Primitive Hematopoietic Cells.
Previous studies have shown that the microenvironment provided by the Stromal cells is crucial in maintaining haematopoiesis for up to 8weeks (Moore et al. 1997). Although the precise mechanism remains unclear, previously McSorley and Daly (2000) showed that CYP enzymes inhibition significantly reduced RA degradation while Bowles et al (2006) showed that precise regulation of retinoid levels during foetal gonad development provides the molecular cue that specifies germ cell fate. Consequently, Ghiaur et al hypothesised that bone marrow may maintain haematopoietic cells in the same way. Thus, they used RA-responsive luciferase reporter assay to evaluate the effect of stroma on RA activity. In this assay, a pGL2-RA responsive element (RARE)-luciferase was transfected into HEK293T cells via Renilla plasmid. Luciferase activity is normally induced by serum, which contains micromolar concentrations of retinoids and can be used to quantify RA. Thus, Luciferase activity decreased significantly when RAR was inhibited with AGN or by cotransfection with a dominant-negative RAR vector, and in the presence of bone marrow-derived stroma. But in the presence of CYP inhibitor, the protective effect of stromal was abolished (fig 4).

Fig 4. Effects of stromal CYP26 on RA metabolism. Quantifying RA activity using RA response element-luciferase reporter shows that inhibiting stroma and or CYP increases the concentration of RA as reported by luciferase activity.
Following this, Ghiaur et al found that bone marrow-derived stroma controls primitive hematopoietic cell fate by probably modulating RA bioavailability through expression of the P450 retinoid hydroxylases- CYP26A1 and CYP26B1. The quantification of CYP26 isoenzymes was assessed by Quantitative RT-PCR using sequence-specific primers. The involvement of the CYP26 isoenzymes was evident by the accelerated loss of CD34 CD38– cells and CAFCW8 after 21days following treatment with a CYP26 inhibitor; and CYP26B1 knockdown which reversed the protective effects of stroma microenvironment- RA bioavailability (fig 5A). Most importantly, the addition of the CYP26 inhibitor to the stromal coculture blocked the maintenance of SRCs thus reducing engraftment (fig. 5B). Moreover, other studies in osteoblast and endothelial cells have shown the expression of CYP26. For instance, Gao et al (2010) showed that OP-9 cells are bona fide Mesenchymal stem cells capable of differentiating into mesenchymal cells and Ghiaur et al also showed that these cells express the CYP26 isoenzymes. Put together, these findings demonstrate that CYP26-mediated clearance of endogenous RA is at least in part responsible for maintenance of the primitive HSC by bone marrow stroma.

Fig 5. Effect of CYP26 in the maintenance of primitive haematopoietic progenitor cells by bone marrow stroma. a) Shows the effects of stromal CYP26 on the maintenance of CAFCW8 cells during coculture of CD34 CD38− cells with or without CYP26 inhibitor for 21 days. b) Percentage of human engraftment in the bone marrow of individual NSG mice, after transplantation of 2,000 CD34 CD38− cells on primary human bone marrow stroma for 21 days in the presence or absence of CYP26 inhibitor R115866. Black diamonds represent individual mice. The SRC frequency per cultured CD34 CD38− equivalents was 1:1,471 in cultures without CYP inhibitor and <1:21,484 with CYP26 inhibitor, P = 0.01.
Conclusion and Future perspectives
This paper elucidated that primitive HSCs could be intrinsically programmed to undergo RA-mediated differentiation unless prevented from doing so by bone marrow niche CYP26 since upstream members of the RA pathways are constitutively expressed. Thus, Ghiaur et al concluded that modulating RA bioavailability in the bone marrow microenvironment could regulate physiologic HSC fate, and that inhibition of RA signalling may be a potential therapeutic tool for human HSC expansion ex vivo. These findings can be useful since it was done using adult human stem cell and so can be translated and useful in regenerative medicine. However, as stated in the study, RA pathway was one of the top pathways to show coordinate inactivation in the CD34 CD38− compared with CD34 CD38 populations. And it was observed that human engraftment was optimal at day 7, after which there was really no significant difference between mice transplanted with cells treated with AGN or TSF. Thus, this raise a question- could the effect seen on HSC expansion be as a result of the other downregulated pathways? Moreover, since HoxB4, Bmi1, Wnt/β-catenin, Notch, and aryl hydrocarbon receptor antagonists have all been shown to promote HSC expansion, concerted inhibition of these pathways is worth elucidating and could be beneficial clinically.
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