Long noncoding RNAs: Emerging regulators of normal and malignant hematopoiesis

: Genome wide analyses have revealed that long-noncoding RNAs (lncRNAs) are not only passive transcription products, but also major regulators of genome structure and transcription. In particular, lncRNAs exert profound effects on various biological processes, such as chromatin structure, transcription, RNA stability and translation, and protein degradation and localization, which depend on their localization and interacting partners. Recent studies have revealed that thousands of lncRNAs are aberrantly expressed in various cancer types and some of them are associated with malignant transformation. Despite extensive efforts, the diverse functions of lncRNAs and molecular mechanisms in which they act remain elusive. Many hematological disorders and malignancies are primarily resulted from genetic alterations that lead to the dysregulation of gene regulatory networks required for cellular proliferation and differentiation. Consequently, a growing list of lncRNAs has been reported for their involvement in the modulation of hematopoietic gene expression networks and hematopoietic stem and progenitor cell (HS/PC) function. Dysregulation of some of these lncRNAs has been attributed to pathogenesis of hematological malignancies. In this review, we will summarize current advances and knowledge of lncRNAs in gene regulation, focusing on the recent progresses on the role of lncRNAs in CTCF/cohesin mediated three-dimensional (3D) genome organization, and how such genome folding signals in turn regulate transcription, HS/PC function and transformation. The knowledge will provide mechanistic and translational insights into HS/PC biology and myeloid malignancy pathophysiology. Abstract: 21 Genome wide analyses have revealed that long-noncoding RNAs (lncRNAs) are not only 22 passive transcription products, but also major regulators of genome structure and transcription. 23 In particular, lncRNAs exert profound effects on various biological processes, such as chromatin 24 structure, transcription, RNA stability and translation, and protein degradation and localization, 25 which depend on their localization and interacting partners. Recent studies have revealed that 26 thousands of lncRNAs are aberrantly expressed in various cancer types and some of them are 27 associated with malignant transformation. Despite extensive efforts, the diverse functions of 28 lncRNAs and molecular mechanisms in which they act remain elusive. Many hematological 29 disorders and malignancies are primarily resulted from genetic alterations that lead to the 30 dysregulation of gene regulatory networks required for cellular proliferation and differentiation. 31 Consequently, a growing list of lncRNAs has been reported for their involvement in the 32 modulation of hematopoietic gene expression networks and hematopoietic stem and progenitor 33 cell (HS/PC) function. Dysregulation of some of these lncRNAs has been attributed to 34 pathogenesis of hematological malignancies. In this review, we will summarize current 35 advances and knowledge of lncRNAs in gene regulation, focusing on the recent progresses on the role of lncRNAs in CTCF/cohesin mediated three-dimensional (3D) genome organization, 37 and how such genome folding signals in turn regulate transcription, HS/PC function and 38 transformation. The knowledge will provide mechanistic and translational insights into HS/PC 39 biology and myeloid malignancy pathophysiology.

passive transcription products, but also major regulators of genome structure and transcription. 23 In particular, lncRNAs exert profound effects on various biological processes, such as chromatin 24 structure, transcription, RNA stability and translation, and protein degradation and localization, 25 which depend on their localization and interacting partners. Recent studies have revealed that 26 thousands of lncRNAs are aberrantly expressed in various cancer types and some of them are 27 associated with malignant transformation. Despite extensive efforts, the diverse functions of 28 lncRNAs and molecular mechanisms in which they act remain elusive. Many hematological 29 disorders and malignancies are primarily resulted from genetic alterations that lead to the 30 dysregulation of gene regulatory networks required for cellular proliferation and differentiation. 31 Consequently, a growing list of lncRNAs has been reported for their involvement in the 32 modulation of hematopoietic gene expression networks and hematopoietic stem and progenitor 33 cell (HS/PC) function. Dysregulation of some of these lncRNAs has been attributed to 34 pathogenesis of hematological malignancies. In this review, we will summarize current 35 advances and knowledge of lncRNAs in gene regulation, focusing on the recent progresses on 36

Introduction 42
One of the most compelling findings of the genome era of research is that the genome is 43 extensive transcribed from non-coding regions and these noncoding transcripts possess 44 potential regulatory function 1,2 . Non-coding transcripts include long non-coding RNAs 45 (lncRNAs), which are >200 nucleotides in length with no or very little protein coding ability, and 46 other distinct classes of non-coding RNAs including microRNAs (miRNA), small nuclear RNAs 47 (snRNAs), and small nucleolar RNAs (snoRNAs). Depending on their locations, lncRNAs can 48 function in the nucleus, nucleolus, and cytoplasm. This review will focus on the nuclear function 49 of several newly emerged lncRNAs in respect to the CTCF/cohesin associated genomic 50 structural and transcriptional regulation in hematopoiesis and leukemogenesis. 51 Many functions of lncRNAs has been uncovered to involve in diverse biological 52 processes, such as imprinting, X chromosome inactivation, apoptosis, cellular proliferation and 53 differentiation, stem cell biology, and tumorigenesis/leukemogenesis 3-8 . The mode of lncRNAs 54 in transcriptional regulation involves in recruitment or decoy of epigenetic regulators, 55 transcription factors, and accessory proteins. However, how and where lncRNAs exert these 56 diverse functions, including whom they interact with, have become very important questions and 57 an intensive research area in order to gain mechanistic insights into this class of RNA 58 molecules. Acute myeloid leukemia (AML) is a heterogeneous disease with genetic or 59 epigenetic alterations that systematically alter gene regulatory networks required for 60 hematopoietic stem and progenitor cell (HS/PC) proliferation and differentiation. A growing list of 61 lncRNAs involved in controlling hematopoietic gene expression networks has been discovered 62 and some of which linked to the dysregulation of HS/PC function in AML 9,10 . In this review, we 63 will focus on molecular mechanisms by which lncRNAs systematically regulate hematopoietic 64 transcription pathways via their specific roles in 3D hematopoietic/leukemic genome 65 organization. 66 67

Overview of mechanisms by which lncRNAs regulate gene transcription. 68
In the nucleus, lncRNAs can regulate gene transcription in both cis and trans actions via 69 physical interactions with target DNA and associated proteins. LncRNAs can regulate epigenetic 70 landscape, enhancer/promoter activity, and RNA polymerase II (RNAPII) machinery by diverse 71 mechanisms 11,12 . 72

A. Scaffold 73
Xist lncRNA-mediated X chromosome inactivation is a well-known example of lncRNA-74 driven transcriptional silencing ( Figure 1A), one of the critical mechanisms leading to the gene 75 dosage compensation in female cells. As a key regulator of this process, lncRNA Xist, through 76 orchestrating 3D chromatin conformation, spreads and coats polycomb repressive complex 2 77 (PRC2) and resulting H3K27me3 modifications on the future inactive X chromosome for stably 78 silencing of this entire X-chromosome 5,13 . It is interesting to note that deletion of Xist in 79 hematopoietic compartment of female mice impaired HSC function and mutant females 80 developed aggressive myeloproliferative neoplasm (MPN) and myelodysplastic syndrome 81 (MDS), which resulted from dysregulated myelo-erythroid fate decision of HSCs 14 . Notably, X-82 linked master erythroid transcription factor, Gata-1, was upregulated in Xist -/females, perhaps 83 due to X-linked gene over-dosage in mutant females 14  remodeling complexes to specific genomic loci. We recently discovered and cloned a unique 98 HoxB locus-associated lncRNA, HoxBlinc, which is expressed during early hematopoietic 99 differentiation and is strongly concomitant with the H3K4me3 patterning and anterior Hoxb gene 100 activation. HoxBlinc associates with the Setd1a/MLL1 histone methyltransferase (HMT) complex 101 to coordinate activation of the anterior Hoxb genes and specify hematopoietic cell fates during 102 early hematopoietic differentiation 3 . It is clear that lncRNAs can act as epigenetic regulators to 103 modulate gene transcription. 104

C. Stabilization 105
Transcription regulation is central in controlling cellular proliferation and differentiation. 106 Many proteins known to be involved in eukaryotic transcription not only can interact with DNA 107 elements, but also involve in RNA interaction. A significant number of transcription factors (TFs) 108 can be modulated by specific lncRNAs and their interaction may stabilize TF in chromatin 109 template ( Figure 1C). One example of such lncRNA is PVT1, a lncRNA required by MYC in "decoy molecule". The lncRNA, growth arrest-specific 5 (Gas5), is involved in starvation-119 associated repression of the glucocorticoid receptor (GR)-mediated transcription 23 . GAS5 120 employed a decoy mechanism to sequester GR from the glucocorticoid responsive genes by the 121 formation of an RNA motif in one of its stem-loop secondary structure that mimics the hormone 122 response element like DNA motif. Therefore, GAS5 competes for the binding of GR to the 123 glucocorticoid response elements in the promoters of glucocorticoid responsive genes 23 ( Figure  124 1D). 125

E. Bridge 126
Enhancer RNAs (eRNAs) are a specific class of lncRNAs that are transcribed from 127 enhancer DNA sequences and may have a profound impact on gene transcription. The p53-128 associated eRNAs were transcribed from the p53-bound enhancer regions (p53BER) that 129 induce p53-independent intra-chromosomal interaction bringing p53BERs into close proximity of 130 the p53 target genes for efficient transcription enhancement of the associated p53 target genes 131 leading to a p53-dependent cell-cycle arrest 24 ( Figure 1E).   (Table 1). One excellent example that lncRNAs serving as a driving force of large-175 scale chromosome organization is the Xist lncRNA-driven X-chromosome inactivation 176 throughout the female life to equalize X-linked gene dosage with male. While Xist initiates and 177 spreads PRC2 and resulting H3K27me3 accumulation along X-chromosome by orchestrating a 178 3D genome topology 5 , Xist also targets CCCTC binding factor (CTCF) to specific genome loci 179 to mediate long-range chromosomal interactions in a locus specific manner during X 180 chromosome inactivation 33 . Mechanistically, Xist lncRNA actively repels cohesin complex from 181 inactive X-chromosome as deletion of Xist restores cohesin binding resulting in reorganization 182 of TADs that resemble active X chromosome 34 , suggesting a central role for lncRNA in the 3D 183 organization of mammalian genome, perhaps mediated by CTCF and its associated cohesin 184 complex. 185 The notion that lncRNAs are involved in CTCF-mediated genome organization stems 186 from a seminal finding by Dr. Gary Felsenfeld's group that DEAD-box RNA helicase p68 and 187 steroid receptor RNA activator (SRA) interact with both CTCF and cohesin. Depletion of p68 or 188 SRA impaired chromatin insulator activity by disrupting CTCF/cohesin interaction 35

Role of lncRNAs in hematopoietic stem cell (HSC) biology. 203
Compared with mRNA, the expression levels of lncRNAs are relatively low, yet the 204 expression of many lncRNAs is highly cell-type/lineage, differentiation stage, or even disease 205 state specific 39-41 , suggesting that lncRNAs act as determinants for lineage commitment and HSCs via aberrant binding of HOTTIP to hematopoietic/leukemic specific TFs or CTCF motifs 7 . 249 In addition to those lncRNAs involved in HSC self-renewal and differentiation, maternally 250 but not paternally deletion of H19 lncRNA and its associated upstream differentially methylated 251 region (DMR) reduced adult HSC quiescence, which play a key role in long-term HSC 252 maintenance. The expression of H19 is maternally imprinted (expressed exclusively from 253 maternal inherited allele) that is regulated by binding of CTCF to the unmethylated DMR. Loss 254 of maternally expressed H19 upregulated paternally associated Igf2 gene pathway that resulted 255 in the release of FoxO3-mediated cell cycle arrest 6 . 256 257

Involvement of HOX-associated lncRNAs in AML leukemic genome organization. 258
AML is a heterogeneous hematopoietic stem or progenitor cell disease that resulted 259 from genetic alterations or somatic mutations of genes required for HS/PC biology. Given that which mechanisms lncRNAs such as HOTTIP are targeted to and/or recognize specific CBSs. It 306 is also important to determine how RNAs are widely required for CTCF-mediated genome 307 organization and molecular basis by which lncRNAs act on CTCF-mediated topological genome 308 regulation. Unbiased isolation of HOTTIP associated protein complex in AML cells would 309 provide mechanistic insight into its action in CTCF mediated genome organization. 310

LncRNAs and their pathways serve as biomarkers and potential therapeutic targets for 312 leukemia 313
Many lncRNAs exhibit lineage-, differentiation stage-, or disease-specific expression, 314 making them excellent candidates for biomarkers or therapeutic applications. A study evaluated 315 the lncRNA expression profiles from 148 untreated older AML patients with normal cytogenetics 316 to determine whether lncRNAs are associated with clinical features and certain recurrent 317 mutations in elderly AML patients. Interestingly, distinct lncRNA expression profiles were highly 318 associated with specific mutations, such as FLT3-ITD, NPM1, CEBPA, IDH2, ASXL1, and 319 RUNX1 genes, while another subset of lncRNAs are highly correlated with treatment response 320 and survival 56 . Recently, through RNA sequencing followed by univariate and multivariate time-321 to-event analysis of 274 AML patients after intensive chemotherapy from a Swedish cohort, 33 322 lncRNAs were found to be associated with overall survival of AML patients and their expression 323 profiles predicted prognostic outcomes of AML patients 57 . These lncRNAs likely play oncogenic 324 roles via their impact on one or several hallmarks of leukemia including self-renewal, anti-325 apoptosis, proliferation, and differentiation. The importance of some of these lncRNAs in 326 reshaping the epigenetic landscape of leukemic genome is beginning to be recognized. These 327 AML-associated specific characteristics of lncRNAs make them potential markers for clinical 328 prognostic outcomes and potential targets for treatment. 329 HOTTIP and HOXBLINC are HOX-associated oncogenic lncRNAs that are aberrantly 330 activated in AML patients carrying MLL rearrangement (MLLr + ) and/or NPM1 C+ mutation 7,45 . 331 HOTTIP lncRNA activation promotes HSC/LSC self-renewal and AML progression by activating 332 HOXA9 and canonical Wnt/-catenin pathway 7 , which is uniquely required for LSC self-renewal 333 interference of interactions between lncRNAs and genetic elements or regulatory proteins at 348 specific loci may be able to block specific leukemic transcription programs (e.g. HOXA9 or -349 catenin), leading to efficient targeted therapy 350 351

Summary and perspectives 352
Novel technologies has been extensively employed in genome research to interrogate 353 the genetic targets and associated protein complexes of specific lncRNAs in cells. Thereby, the 354 actions of lncRNAs in lineage-or disease-specific genome organization broadened in the past 355 several years. However, the detailed mechanisms involved in lncRNA dependent genome 356 topology remains to be explored. Although HOTTIP lncRNA is shown to mediate TAD formation 357 in several critical hematopoietic/leukemogenic loci during AML progression 7 , yet many 358 questions remain unanswered. Mechanistically, it remains unclear how lncRNA regulates TAD 359 and TAD boundaries. It remains to be determined whether and how lncRNAs recruit, bridge, 360 guide, or facilitate the CTCF/cohesin complex to specific genomic regions to alter genome 361 topological structure. CTCF is a master regulator of mammalian genome organization 60 . CTCF 362 acts to modulate chromatin TAD boundary, as well as enhancer/promoter contacts within TAD 363 in cohesin-dependent or -independent manners 61,62 . Although CTCF core motif is highly 364 enriched in HOTTIP-bound genomic regions 7 , it remains to be determined whether lncRNAs 365 directly regulate the ability of CTCF to access chromatin DNA or its association with the cohesin 366 complex. Cohesin complex is frequently colocalized with CTCF in the genome and their binding 367 sites are mutational hotspots in the noncoding cancer genome 63 . In addition, the cohesin genes 368 are frequently mutated in AML that exhibits a strong association with NPM1 mutation 64 . 369 Therefore, it appears that CTCF/cohesin mediated function plays a critical role in HS/PC 370 regulation and AML leukemogenesis, perhaps through their actions in hematopoietic specific 371 genome topology. Recent report has shown that architectural RNA is essential for PRC2 372 complex chromatin occupancy 15 , suggesting that RNA provide an additional regulatory layer 373 and may have a wide role in chromatin domain organization beyond PRC2-repressive complex.

Conflict-of-interest disclosure: 405
The authors have declared that no competing interests exist. 406