Transcriptomes reflect the phenotypes of undifferentiated, granulocyte and macrophage forms of HL-60/S4 cells

Abstract

To understand the chromatin changes underlying differential gene expression during induced differentiation of human leukemic HL-60/S4 cells, we conducted RNA-Seq analysis on quadruplicate cultures of undifferentiated, granulocytic-and macrophage-differentiated cell forms. More than half of mapped genes exhibited altered transcript levels in the differentiated cell forms. In general, more genes showed increased mRNA levels in the granulocytic form and in the macrophage form, than showed decreased levels. The majority of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were significantly enriched in genes that exhibited differential transcript levels after either RA or TPA treatment. Changes in transcript levels for groups of genes with characteristic protein phenotypes, such as genes encoding cytoplasmic granular proteins, nuclear envelope and cytoskeletal proteins, cell adhesion proteins, and proteins involved in the cell cycle and apoptosis illustrate the profound differences among the various cell states. In addition to the transcriptome analyses, companion karyotyping by M-FISH of undifferentiated HL-60/S4 cells revealed a plethora of chromosome alterations, compared with normal human cells. The present mRNA profiling provides important information related to nuclear shape changes (e.g., granulocyte lobulation), deformability of the nuclear envelope and linkage between the nuclear envelope and cytoskeleton during induced myeloid chromatin differentiation.

Hi-C detects novel structural variants in HL-60 and HL-60/S4 cell lines

“…A recent karyotype using FISH reported additional SVs (i.e. inv (2) [22] were detected with hic_breakfinder in the HL-60/S4-HindIII dataset (this study) but not the HL-60/S4-MboI dataset [25] ( Supp table 3). This apparent discrepancy may be due to restriction enzyme choice or other library preparation factors, especially given that both missing regions involve nearby regions of chromosome 16.…”

“…Similarly, we observed high sequence coverage of the six amplified regions in normalized DNase hypersensitivity data from ENCODE (Fig 2d, [40]). As reported in [22], the HL-60/S4 chromosome 8 is involved in two SVs, ins(6;8)(q22.2;q24.13-q24.21) and der(6;8;11)(q22.2;q24.13-q24.21;p14.1). Hi-C data of HL-60/S4 confirms that the MYC expansion is involved in translocations with chromosomes 6 and 11 (Fig 2b, Supp fig 2b).…”

“…Inversions on chromosomes 20(p11.23;q11.23) were identified with hic_breakfinder within the HL-60/S4-MboI Hi-C dataset, while an inversion on chromosome 17(q21.1;q21.32) was detected in HL-60/S4-HindIII Hi-C dataset (Supp fig 4d,e). A large pericentric inversion on chromosome 2(q22.1;q31.2) was reported in the HL-60/S4 reference karyotype [22], however, we detected four additional shorter-range SVs within chromosome 2 (i.e. t(2;2)(p24.2;p13.1), inv(2)(p15;p13.1) (Fig 3a, Supp fig 3a,b), del(2)(q14.1-q22.1), inv(2)(q22.3;q24.3) (Supp fig 4a,b))( Table 1).…”

“…The next two columns indicate whether the SV was detected in HL-60/CICLR or HL-60/S4 respectively. A starred "yes*" indicates that this SV is not reported in the reference karyotype (DSMZ for HL-60/CICLR and Welch 2017 [22] for HL-60/S4). The Fusion Gene column indicates whether a fusion gene was identified at the SV.…”

“…While HL-60 and HL-60/S4 are recognized as different cell lines by ATCC, they are both used for similar studies [22,24]. It is notable that despite the HL-60/S4 variant being developed as a super-responder to retinoic acid-induced differentiation; modern studies typically treat both HL-60 and HL-60/S4 with 1µM retinoic acid for four days to induce differentiation into the granulocytic form [22,24].…”